JP2016166184A - Compositions and methods for detecting egfr mutations in cancer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide binding agents to an E746-A750 deletion and an L858R point mutation in an epidermal growth factor receptor (EGFR) molecule, and methods for use thereof, including methods for diagnosis and treatment of cancer.SOLUTION: A binding agent specifically binds to an epidermal growth factor receptor (EGFR) molecule comprising a deletion at position E746-A750 and to an EGFR molecule comprising an L858R point mutation, and is, e.g., a rabbit monoclonal antibody or the like.SELECTED DRAWING: Figure 1

Description

This application claims the benefit and priority of US provisional application USSN 61 / 123,699 filed on April 10, 2008 and US provisional application USSN 61 / 190,597 filed on August 29, 2008. The disclosures of both such applications are hereby incorporated by reference in their entirety.

  The present invention relates generally to the field of mutant proteins and genes involved in cancer, as well as cancer detection, diagnosis and therapy.

  Cancer is the main cause of human death. Lung cancer is the leading cause of cancer-related deaths worldwide and is expected to remain a major health challenge. Lung cancer is roughly classified into small cell lung cancer (SCLC, 20% of lung cancer) and non-small cell lung cancer (NSCLC, 80% of lung cancer). Somatic mutations in the epidermal growth factor receptor (EGFR) gene have been found in a subgroup of lung adenocarcinoma, and EGFR tyrosine kinase inhibitor (TKI) gefitinib [Lynch, TJ et al., N Engl J Med, 2004. 350 (21): p.2129-39 and Paez, JG et al., Science, 2004. 304 (5676): p.1497-500] and erlotinib [Pao, W. et al., Proc Natl Acad Sci USA, 2004. 101 ( 36): associated with sensitivity to p.13306-11]. Although many types of EGFR mutations have been reported, the most common non-small cell lung cancer (NSCLC) -related EGFR mutations are exon 19 15 bp nucleotide in-frame deletions (E746-A750del) and exon 21 codons Point mutation (L858R) in which 858 leucine is replaced with arginine [Pao, W. et al., Proc Natl Acad Sci USA, 2004. 101 (36): p.13306-11; Riely, GJ et al., Clin Cancer Res, 2006. 12 (24): p.7232-41; and Kosaka, T. et al., Cancer Res, 2004. 64 (24): p.8919-23]. These two mutations represent 85-90% of EGFR mutations in NSCLC patients. Importantly, these mutation carriers have been shown to respond well to EGFR inhibitors such as gefitinib and erlotinib [Riely, GJ et al., Clin Cancer Res, 2006. 12 (24): p.7232-41; Inoue, A. et al., J Clin Oncol, 2006. 24 (21): p.3340-6; Marchetti, A. et al., J Clin Oncol, 2005. 23 (4): p.857- 65; and Mitsudomi, T. et al., J Clin Oncol, 2005. 23 (11): p.2513-20.]. Therefore, detection of these mutations is one important way to improve the treatment of patients with lung cancer.

  PCR amplification to detect EGFR mutations in the patient's tumor tissue because EGFR mutation analysis in lung adenocarcinoma can guide treatment decisions and enroll patients in specific treatment groups in clinical trials Direct DNA sequencing of the product has been developed. However, these tests have not been widely adopted due to expensive equipment and reagents, difficulty in performing the assay, and the time required to complete the test. In addition, DNA sequencing has limited sensitivity to detect tumor cells with EGFR mutations in the background of normal cells without the mutant. A minimum of 50% tumor cells are required to ensure EGFR sequencing assay accuracy. Recently, other DNA-based methods have been developed to improve the detection of EGFR mutations in lung cancer specimens, such as genotyping based on TaqMan PCR, Scorpions ARMS, MALDI TOF MS, dHPLC, and single molecule sequencing. Yes. However, these methods are not routine procedures in clinical laboratories and are still expensive and require a long time. Also, cell-based mutation states are not identified. Thus, the sensitivity of these methods depends on the tumor cell rate in the sample used for homogenate production, and samples obtained from standard biopsies are usually insufficient for DNA sequencing. On the other hand, immunohistochemistry (IHC) is a well-established solid tumor analysis method routinely practiced in all clinical laboratories. This method is a more accessible technique in clinical diagnosis, and the rate of cancer cells or the amount of tumor tissue in tumor specimens available for analysis has little impact on interpretation. The method also allows simultaneous analysis of other proteins or protein modifications. However, the total expression level of EGFR by IHC has not been observed to predict the response of therapy with tyrosine kinase inhibitors of NSCLC [Meert, AP et al., Eur Respir J, 2002. 20 (4): p.975 -81]. Therefore, the development of antibodies that specifically detect mutant EGFR proteins that can be used for IHC would be an added value in clinical diagnosis and treatment of lung cancer.

  A related challenge faced by diagnostic analysis of solid tumor samples such as lung cancer tumors is access to tissue samples. Repeated biopsies are clinically not feasible in almost all tumor types. Therefore, alternative cancer cell sources must be obtained. This is particularly important in the context of targeted therapy in repeated tumor analysis that can be used to guide drug therapy. Cancer tissue types of circulating tumor cells (CTC) selected from the group consisting of lung cancer, colon cancer, breast cancer, cervical cancer, pancreatic cancer, prostate cancer, gastric cancer, and esophageal cancer, ascites, tracheal swab, ductal adenocarcinoma, etc. In some tumor types, several cancer cell sources are available. Circulating proteins can be detected by standard protein assays such as ELISA assays. In this example, the mutant EGFR protein is captured and detected by a pair of antibodies including an antibody against the total protein and an antibody against the mutation. Such assays allow for routine and iterative analysis of treated patients to select optimal drugs and drug regimens that directly affect the patient's tumor.

  The present invention provides a binding agent (eg, a rabbit monoclonal antibody) that specifically binds to an E746-A750-deficient EGFR molecule and an EGFR molecule having an L858R point mutation.

  Accordingly, in the first aspect of the present invention, a binding agent that specifically binds to an epidermal growth factor receptor (EGFR) molecule that is deficient in the E746-A750 position is provided. In some embodiments, the epidermal growth factor receptor (EGFR) molecule is from a human. In some embodiments, the binding agent comprises at least one complementarity determining region (CDR), wherein the CDR is SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: Contains a sequence selected from the group consisting of 18. In some embodiments, the binding agent specifically binds to an epitope comprising an amino acid sequence comprising a threonine-serine-proline sequence. In some embodiments where the binding agent is an antibody, the antibody is produced by a clone deposited with the ATCC and is assigned ATCC designation number PTA-9151.

  In another aspect of the invention, a binding agent is provided that specifically binds to an epidermal growth factor receptor (EGFR) molecule having a point mutation in which leucine at position 858 is replaced with arginine. In some embodiments, the epidermal growth factor receptor (EGFR) molecule is from a human. In some embodiments, the binding agent comprises at least one complementarity determining region (CDR), the CDR comprising SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 31, and sequence Comprising an amino acid sequence selected from the group consisting of number 32. In some embodiments, the binding agent specifically binds to an epitope comprising an amino acid sequence comprising a threonine-aspartate-X-glycine-arginine sequence (where X is any amino acid residue). In some embodiments where the binding agent is an antibody, the antibody is produced by a clone deposited with the ATCC and is assigned ATCC designation number PTA-9152.

  In a further aspect of the invention, a polynucleotide (eg, a purified polynucleotide) is provided that encodes a binding agent that specifically binds to an epidermal growth factor receptor (EGFR) molecule that is defective at position E746-A750. In a further aspect of the invention, a polynucleotide encoding a binding agent that specifically binds to an epidermal growth factor receptor (EGFR) molecule having a point mutation in which leucine at position 858 is replaced with arginine (e.g., purified polynucleotide Nucleotides). In a further aspect of the invention, a vector (eg, an expression vector) containing the polynucleotide is provided.

  In another aspect of the present invention, a method for identifying cancer in which targeted therapy for abnormal expression of EGFR molecules is effective. The method comprises the steps of: (a) contacting a biological sample derived from cancer with a binding agent that specifically binds to an epidermal growth factor receptor (EGFR) molecule lacking E746-A750 position to obtain a binding amount; (B) comparing the result of the step (a) with the amount of binding obtained by contacting a biological sample derived from a healthy individual with a binding agent, and the difference between the amount of binding derived from a healthy individual and the amount of binding derived from a cancer. And the step of showing the amount of binding that the therapy responds well to the cancer. In various embodiments, the tissue type of a cancer-derived biological sample and a healthy individual-derived biological sample are the same. In some embodiments, the cancer is from a human patient. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the cancer is adenocarcinoma or squamous cell carcinoma. In some embodiments, the cancer is a tissue type selected from the group consisting of lung cancer, colon cancer, breast cancer, cervical cancer, pancreatic cancer, prostate cancer, gastric cancer, and esophageal cancer.

  In another aspect of the present invention, a method for identifying cancer in which targeted therapy for abnormal expression of EGFR molecules is effective. The method comprises (a) contacting a cancer-derived biological sample with a binding agent that specifically binds to an epidermal growth factor receptor (EGFR) molecule having a point mutation in which leucine at position 858 is replaced with arginine. A step of obtaining a binding amount, and (b) comparing the result of the step (a) with a binding amount obtained by bringing a biological sample derived from a healthy individual into contact with a binding agent. And the step of showing the amount of binding that the therapy responds well to the cancer due to the difference from the amount of binding. In various embodiments, the tissue type of a cancer-derived biological sample and a healthy individual-derived biological sample are the same. In some embodiments, the cancer is from a human patient. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the cancer is an adenocarcinoma. In some embodiments, the adenocarcinoma is a tissue type cancer selected from the group consisting of lung cancer, colon cancer, breast cancer, cervical cancer, pancreatic cancer, prostate cancer, gastric cancer, and esophageal cancer.

  In various embodiments, the amount of binding is determined using an assay method selected from the group consisting of Western blot, immunofluorescence, ELISA, IHC, flow cytometry, immunoprecipitation, autoradiography, scintillation counter, and chromatography. To do.

  In a further aspect of the invention, a binding agent that specifically binds to an epidermal growth factor receptor (EGFR) molecule having a point mutation in which leucine at position 858 is replaced by arginine, an epithelium lacking position E746-A750 Also provided are binding agents that specifically bind to growth factor receptor (EGFR) molecules, or compositions that contain both binding agents. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. The present invention relates to a polynucleotide encoding a binding agent that specifically binds to an epidermal growth factor receptor (EGFR) molecule having a point mutation in which leucine at position 858 is replaced by arginine, and lacks E746-A750 position. Also provided is a polynucleotide encoding a binding agent that specifically binds to an epidermal growth factor receptor (EGFR) molecule, or a composition containing both polynucleotides. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

  In a further aspect of the present invention, there is provided a method for treating a patient or patient with cancer, or a patient or patient suspected of having the cancer, in which targeted therapy for abnormal expression of EGFR molecules is effective. The method includes administering an effective amount of a composition of the invention to a patient or patient.

  In another aspect of the invention, a method for identifying L858R point mutations and / or E746-A750 deficiency in the EGFR status of a patient or patient is disclosed, the method comprising: a) obtaining a biological sample from the patient or patient; b) screening the sample with a binding agent that specifically binds to an L858R point mutation in EGFR and / or E746-A750 deletion, and c) an E746-A750 deletion and / or L8585R point mutation in EGFR in the sample. Determining whether or not. In some embodiments, the method comprises screening a sample with a wild type EGFR specific antibody. In some embodiments, the method comprises screening a sample with a pan keratin antibody (eg, a pan cytokeratin antibody).

  In another embodiment of the present invention, a detection kit for detecting E746-A750 deletion or L858R point mutation in EGFR in a sample is described, the kit comprising (a) binding that specifically binds to E746-A750 deletion in EGFR An agent and / or a binding agent that specifically binds to an L858R point mutation in EGFR; and b) an instruction to detect an E746-A750 deficient or L858R point mutation in EGFR in a sample.

FIG. 3 is a representative Western blot showing the reactivity of the antibodies of the invention against EGFR and its mutants in a specified cell line. Control wild-type (wt) EGFR-specific antibody clone 86 (upper panel) is somewhat less reactive in mutant EGFR-expressing cells (i.e., HCC827, H1975, H3255, and H1650 cells) Bind to (ie, react to) lysates prepared from all cell lines. EGFR L858R specific antibody (clone 6B6) reacts only with H175 and H3255 cells (middle panel), whereas dEGFR (i.e. EGFR del746-A750) specific antibody (clone 43B2) reacts only with HCC827 and H1650 cells To do. FIG. 3 shows the reactivity of the antibodies of the present invention against EGFR and its mutants in a specified cell line by immunofluorescence cytochemistry. The control EGFR specific antibody (upper panel) stains all 6 cell lines (ie, binds to all 6 cell lines) regardless of EGFR mutation status. EGFR L858R-specific antibodies stain only cancer cells that have an L858R point mutation of the EGFR molecule. Similarly, dEGFR-specific antibodies stain only cancer cells that are deficient in exon 19 (ie, E746-A750) in the EGFR molecule. The reactivity by the immunohistochemical method of the antibody of this invention with respect to EGFR and its mutant in the section | slice derived from the nude mouse which transplanted the designated cell line as a xenograft is shown. The control EGFR specific antibody (upper panel) stains all 6 cell lines (ie, binds to all 6 cell lines) regardless of EGFR mutation status. EGFR L858R-specific antibodies stain only cancer cells that have an L858R point mutation of the EGFR molecule. Similarly, dEGFR-specific antibodies stain only cancer cells that are deficient in EGFR intramolecular exon 19 (ie, E746-A750). FIG. 4 shows the reactivity of the antibodies of the present invention by 4 representative, non-limiting, NSCLC samples before genotyping (ie, samples whose DNA sequence has not been determined prior to IHC analysis). Samples from patients CL109 and CL745 that were known to carry EGFR L858R point mutations by DNA sequencing were positive for L858R-specific antibody staining but negative for dEGFR-specific antibody staining. there were. Samples from patients CL495 and CL712, known to be deficient in E746-A750 by DNA sequencing, were positive for dEGFR-specific antibodies but negative for L858R-specific antibodies. FIG. 6 shows the immunohistochemical reactivity of the antibodies of the present invention against two representative, non-limiting, genotyped NSCLC samples (ie, samples whose DNA sequence has not been determined prior to IHC analysis). Tumor samples from patient CL761 were positive for pancytokeratin-specific antibody, control wild-type EGFR-specific antibody, and L858R-specific antibody staining but negative for dEGFR (ie, E746-A750del) -specific antibody staining Met. In contrast, tumor samples from patient CL764 were positive for pancytokeratin specific antibody (positive control), control wild type EGFR specific antibody and dEGFR specific antibody staining but negative for L858R specific antibody. there were.

  The present invention relates generally to mutant proteins and genes involved in cancer, and cancer detection, diagnosis and treatment using the antibodies of the present invention disclosed herein.

  Immunohistochemistry showed high EGFR protein expression in the majority of squamous cell carcinomas, but low expression in large cell, adenocarcinoma, and prebronchial lesions, suggesting significance in lung carcinogenesis [Selvaggi, G. et al., Ann Oncol, 2004. 15 (1): p.28-32]. There are conflicting data on the importance of prognostic prediction of EGFR protein concentration in NSCLC. A meta-analysis of these studies showed no significant correlation between EGFR levels and survival [Meert, A.P. et al., Eur Respir J, 2002. 20 (4): p.975-81]. In an original study of gefitinib and later study data, retrospective assessment of the association between EGFR positivity and response in immunohistochemistry showed that EGFR immunohistochemistry results did not predict response [Clark, GM J Thorac Oncol, 2006. 1 (8): p.837-46; Tsao, MS et al., N Engl J Med, 2005. 353 (2): p.133-44; Dziadziuszko, R. et al., Ann Oncol , 2007. 18 (3): p.447-52; and Cappuzzo, F. et al., J Natl Cancer Inst, 2005. 97 (9): p.643-55]. Because the presence of certain EGFR mutations correlates with the clinical response of either gefitinib or erlotinib, there is a high demand for the identification of such EGFR mutations in NSCLC patients.

  Accordingly, the present invention provides rabbit mAbs with selective reactivity against the EGFR protein with E746-A750del and L858R point mutations made as described herein. The antibody was shown to be specific for E746-A750del and L858R mutant EGFR protein by Western blot and immunofluorescence. The antibodies in NSCLC xenograft tumors, cell pellets and pre-molecular identification samples were further analyzed by IHC and compared to anti-wtEGFR mAbs. RmAb was selected to detect either E746-A750del or L858R point mutant EGFR protein and not wtEGFR or other types of EGFR mutations. On the other hand, anti-wtEGFRAb reacted extensively with a higher proportion of NSCLC. Thus, the binding agents described herein specifically recognize either E746-A750del or L858R mutant EGFR protein.

  The present invention provides binding agents (such as antibodies) that specifically bind to the EGFR L858R mutation and the EGFR E746-A750del mutation. EGFR mutation-specific antibodies may be used in clinical management (e.g., treatment and diagnosis) of cancer patients or patients, particularly patients or patients with other cancers characterized by NSCLC or EGFR abnormalities, or patients suspected of having the cancer. Very valuable in.

  As used herein, the singular forms “a,” “a,” “an,” and “the,” are used unless the context clearly indicates otherwise. Also specifically included are plural forms derived from the terms they indicate.

  As used herein, the term “about” means roughly, or approximately in the sense of around. Where the term “about” is used in connection with a numerical range, it modifies that range by extending the upper and lower limits of the numerical value set forth. As used herein, the term “about” is typically used to correct numbers above and below the stated values with a difference of 20%.

  Unless otherwise specified, the term “or” as used herein is the “inclusive” meaning of “and / or” and not the “exclusive” meaning of “alternative”. . In this specification and the appended claims, the singular forms also include the plural of the referent unless the context clearly dictates otherwise.

  The terms “including” and “including”, as used herein, whether in a transitional phrase or in the text of a claim, should be interpreted as having an open-ended meaning. That is, the terms are interpreted synonymously with the phrases “having at least” or “including at least”. When used in reference to a process, the term “comprising” means that the process includes at least the listed steps and may include additional steps. As used with respect to a compound or composition, the term “comprising” means that the compound or composition includes at least the listed features or components and may include additional features or components.

  The patents, published applications, and scientific literature referred to herein establish the knowledge of those skilled in the art and are incorporated herein by reference as if each were individually and specifically indicated by reference in their entirety. Incorporated in the specification. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Similarly, any conflict between the art-recognized definition of a word or phrase and the definition of a term or phrase specifically taught herein shall be resolved in favor of the latter. To do.

  Any suitable material and / or method known to those skilled in the art can be used in the practice of the present invention. However, preferred materials and methods are described. Materials, reagents and the like mentioned in the following description and examples are available from commercial sources unless otherwise stated.

  The recitation of a variable range as used herein is meant to indicate that the present invention can be practiced with the same variables as any value included within that range. Thus, if it is essentially a discontinuous variable, the variable can be the same as any integer value including both ends of the numerical range. Similarly, if it is essentially a continuous variable, the variable can be the same as any real value including both ends of the numerical range. As an example, a variable described as a value 0-2 can be 0, 1 or 2 if it is essentially a discontinuous variable, and 0.0, 0.1, 0.01, 0.001 if it is essentially a continuous variable. Or any other real value.

  Hereinafter, specific embodiments of the present invention will be described in detail. While the invention will be described in conjunction with these specific embodiments, it will be understood that it is not intended to limit the invention to such specific embodiments. On the contrary, the intent is to cover alternatives, modifications and equivalents that may be included within the spirit and scope of the invention as defined in the appended claims. In the following description, numerous detailed descriptions are set forth to provide a thorough understanding of the present invention. The present invention may be practiced without some or all of these specific descriptions. In other instances, well known process operations have not been described in detail in order not to unnecessarily obscure the present invention.

  Epidermal growth factor receptors (EGFR; also known as human ErbB-1 and HER1) are receptors on the cell surface of members of the epidermal growth factor family (EGF family) of extracellular protein-based ligands. The amino acid sequence of wild-type human EGFR (including signal sequence) is shown herein as SEQ ID NO: 47, and the amino acid sequence of wild-type human EGFR (without signal sequence) is shown as SEQ ID NO: 48 herein. . Non-small cell lung cancer (NSCLC) patients who carry somatic mutations in epidermal growth factor receptor (EGFR) are treated with the EGFR tyrosine kinase inhibitor gefitinib [Lynch, TJ et al., N Engl J Med, 2004. 350 (21) : p.2129-39 and Paez, JG et al., Science, 2004. 304 (5676): p.1497-500] and erlotinib [Pao, W. et al., Proc Natl Acad Sci USA, 2004. 101 (36): p .13306-11] have been shown to be highly sensitive.

  Mutations are known to occur in the EGFR molecule. As used herein, the term “mutant” or “mutation” refers to a molecule (eg, a polypeptide or polynucleotide) having a structure that is different from a wild-type molecule. Structural differences from wild-type molecules include different sequences (e.g., different amino acid or nucleotide sequences), additional sequences, deleted sequences (i.e., a portion of the sequence is deleted), modification changes (e.g., Methylation, phosphorylation, etc.) and / or fusion of another molecule with all or part of the wild-type molecule, but is not limited thereto. “Wild type” refers to a molecular form that occurs naturally in most individuals of the species from which the mutant molecule is derived, and / or native to healthy individuals of the species from which the mutant molecule is derived (eg, non-neoplastic). Means the molecular form that occurs in Wild type molecular sequences are typically provided in the GenBank database. For example, the amino acid sequence of wild-type human EGFR is shown in SEQ ID NO: 47 (not including a signal sequence having a length of 24 amino acids) and SEQ ID NO: 48 (including a signal sequence).

  As used herein, “EGFR mutant” includes any EGFR intramutation (ie, altered) type that provides an EGFR mutant that differs from wild-type EGFR. The most common NSCLC-related EGFR mutations are in-frame deletions of 15 bp nucleotides within exon 19 (E746-A750del; amino acid sequence shown in SEQ ID NO: 49 (including signal sequence) and amino acids that do not include the signal sequence shown in SEQ ID NO: 50 Sequence) and a point mutation in which leucine at exon 21 codon 858 is replaced with arginine (L858R; amino acid sequence shown in SEQ ID NO: 51 (including signal sequence) and amino acid sequence not including the signal sequence shown in SEQ ID NO: 52). These two EGFR mutants account for 85-90% of EGFR mutations [Riely, G.J. et al., Clin Cancer Res, 2006. 12 (24): p.7232-41]. The ability to detect mutated gene products in cancer cells can identify patients who are most likely to benefit from such treatment, making clinical trials more efficient and beneficial.

  Accordingly, in one aspect of the invention, a binding agent that specifically binds to an epidermal growth factor receptor (EGFR) molecule that is deficient in the E746-A750 position is provided. In some embodiments, the epidermal growth factor receptor (EGFR) molecule is from a human. In some embodiments, the binding agent comprises at least one complementarity determining region (CDR), wherein the CDR is SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: Contains a sequence selected from the group consisting of 18. In some embodiments, the binding agent specifically binds to an epitope comprising an amino acid sequence comprising a threonine-serine-proline sequence.

  In another aspect of the invention, a binding agent is provided that specifically binds to an epidermal growth factor receptor (EGFR) molecule having a point mutation in which leucine at position 858 is replaced with arginine. In some embodiments, the epidermal growth factor receptor (EGFR) molecule is from a human. In some embodiments, the binding agent comprises at least one complementarity determining region (CDR) comprising: SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 31, and sequence Comprising an amino acid sequence selected from the group consisting of number 32. In some embodiments, the binding agent specifically binds to an epitope comprising an amino acid sequence comprising a threonine-aspartate-X-glycine-arginine sequence (where X is any amino acid residue).

  As used herein, a `` binding agent '' is an organic molecule such as a polypeptide (e.g., an antibody as defined herein) or a polynucleotide, or an inorganic molecule such as a lower chemical molecule or synthetic polymer, It means a molecule including, but not limited to, those capable of binding to a reference target molecule (sometimes referred to as an antigen). In some embodiments, the binding agent specifically binds to a reference target molecule. As used herein, “specific binding” or “specifically binds” refers to a binding agent (eg, antibody) of the invention that interacts with its target molecule (eg, an EGFR E746-A750 deletion mutant). Acts and the interaction depends on the presence of a specific structure (i.e., antigenic determinant or epitope) on the target molecule, i.e., the binding agent generally recognizes and binds to the specific structure rather than all molecules Means that. A binding agent that specifically binds to a target molecule can be referred to as a target-specific binding agent. For example, an antibody that specifically binds to an EGFR L858R polypeptide may be referred to as an EGFR L858R specific antibody (or an EGFR L858R variant specific antibody).

  In some embodiments, the binding agents of the invention are purified.

  By “purified” (or “isolated”), a molecule, such as a nucleic acid sequence (eg, polynucleotide) or amino acid sequence (eg, polypeptide), from other components present in the molecule's natural environment Refers to a molecule that has been removed or separated. For example, an isolated antibody is one that is separated from other components of a eukaryotic cell (eg, endoplasmic reticulum or cytoplasmic protein and RNA). A polynucleotide encoding an isolated antibody is one that has been separated from other nuclear components (e.g., histones) and / or upstream or downstream nucleic acid sequences (e.g., a polynucleotide encoding an isolated antibody is May be separated from the endogenous heavy or light chain promoter). An isolated nucleic acid or amino acid sequence of the present invention is at least 60% away from, or at least 75% away from, or at least 90% from other components that naturally accompany the specified nucleic acid or amino acid sequence. May be at least 95% away, or at least 95% away.

  In various embodiments of the invention, the reference target molecule to which the binding agent specifically binds is an EGFR L858R mutant polypeptide (also referred to as a mutation) or an EGFR E746-A750del mutant ( mutant) polypeptide. In some embodiments, the EGFR L858R polypeptide has an amino acid sequence as set forth in SEQ ID NO: 51 or SEQ ID NO: 52. In some embodiments, the EGFR E746-A750del polypeptide has an amino acid sequence as set forth in SEQ ID NO: 49 or SEQ ID NO: 50.

  As used herein, the terms “polypeptide”, “peptide” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched and may contain modified amino acids. Where an amino acid sequence is presented, the sequence is in the N-terminal to C-terminal direction unless otherwise specified (eg, the TSP sequence is N'threonine-serine-proline C '). In some embodiments, the polymer may be interrupted by non-amino acids. The term may also be modified naturally or by intervention (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, e.g., conjugation with a labeling component, etc. It also includes amino acid polymers. Also included in the definition are polypeptides containing, for example, one or more analogs of amino acids (eg, unnatural amino acids, etc.), as well as other modifications known in the art. It will be appreciated that since the polypeptides of the present invention are based on antibodies, the polypeptides may exist as single chains or associated chains.

In some embodiments, the binding agents of the present invention have the following K _D values 1 × 10 ^-6 M to the target molecule (e.g., EGFR L858R polypeptide). In some embodiments, the binding agent of the present invention has a K _D value of 1 × 10 ⁻⁷ M or less, or a K _D value of 1 × 10 ⁻⁸ M or less, or a K _D value of 1 × 10 ⁻⁹ M or less, or ^Binds to a target molecule with a K _D value of 1 × 10 ⁻¹⁰ M or less, a K _D value of 1 × 10 ⁻¹¹ M or less, and a K _D value of 1 × 10 ⁻¹² M or less. In certain embodiments, K _D value of the coupling agent of the present invention is 1pM~500pM or 500PM～1myuM, or 1μM~100nM or 100MM～10nM,,. The term "K _D values" as used herein, the dissociation constant of the interaction between two molecules (such as binding agents (e.g., antibodies) and the dissociation constant between the specific target molecule) intended to refer to a doing.

  In some embodiments, the binding molecule is an antibody.

Natural antibodies (also called immunoglobulins) are composed of two types of polypeptide chains: light chains and heavy chains. The antibody of the present invention may be four intact immunoglobulin chain antibodies including, but not limited to, two heavy chains and two light chains. The antibody heavy chain may be any isotype such as IgM, IgG, IgE, IgG, IgA or IgD, or any subisotype such as IgG1, IgG2, IgG3, IgG4, IgE1, IgE2. The light chain may be a kappa or lambda light chain. One natural antibody has two identical light chains and two identical heavy chains. Heavy chains, each containing one variable region (V _H ) and multiple constant regions, are linked together by intra-constant region disulfide bonds to form the “stem” of the antibody. Each light chain containing one variable region (V _L ) and one constant region is bound to one heavy chain by a disulfide bond. Each light chain variable region is aligned with a bound heavy chain variable region. Both the light and heavy chain variable regions contain three hypervariable regions sandwiched between four highly conserved framework regions (FR). These hypervariable regions, known as complementarity-determining regions (CDRs), form loops that contain antibody-specific antigen binding surfaces (Kabat, EA et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md ., (1987)). Most of the four framework regions have a β-sheet structure, and the CDR forms a loop that connects to the β-sheet structure, and in some cases forms a loop that forms part of the β-sheet structure. Each chain CDR is close to the framework region and contributes to the formation of the antigen binding region together with the CDRs of other chains.

  The present invention also includes antibody components such as single-chain antibodies, antibody molecules of less than four chains such as camelid antibodies, heavy chains or light chains.

Thus, as used herein, the term “antibody” refers to any isotype or subisotype (eg, IgG, IgG1, IgG2a, IgG2b, etc.) from any species (eg, human, rodent, camelid). IgG3, IgG4, IgM, IgD, IgE, IgE1, IgE2, or IgA) intact immunoglobulin molecules, and antigen-binding region fragments thereof such as Fab, Fab ', F (ab') ₂ ; scFv, Fv, Fd, dAb Bispecific scFv, diabody, linear antibodies and other variants thereof (see US Pat. No. 5,641,870, Zapata et al., Protein Eng 8 (10): 1057-1062 [1995]); single chain antibody molecules, And multiple antibodies formed from antibody fragments; and any polypeptide comprising an antibody binding region or a binding region homologous thereto. An `` antigen-binding region '' is any part of an antibody that retains the specific binding activity of an intact antibody (i.e., any part of an antibody that can specifically bind to an epitope on the target molecule of the intact antibody). means. As used herein, the term “epitope” refers to the smallest portion of a target molecule that can be specifically bound by an antigen binding region (eg, of an antibody) of a binding agent. The minimum size of an epitope can be about 5 or 6 to 7 amino acids. Antigen binding regions include, but are not limited to, intact antibody heavy and / or light chain CDR portions, intact antibody heavy and / or light chain variable regions, intact antibody full length heavy or light chain, Or a single CDR of either the heavy or light chain of an intact antibody.

  Antibodies of the present invention include polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies and fragments thereof, and chimeric antibodies comprising an immunoglobulin binding region fused to another polypeptide. It is not limited to.

  The term “does not bind” with respect to a binding agent means that the binding agent (eg, antibody) does not substantially react with the designated molecule. A binding agent (e.g., an EGFR L858R mutation-specific antibody) is compared to another target (e.g., wild-type EGFR) compared to a non-specific control antibody (i.e., does not specifically bind to any molecule). When it is confirmed by a commonly used experimental detection system (Western blotting, IHC, immunofluorescence, etc.) that it does not clearly bind, or when it binds to another target molecule (such as the pancytokeratin specific antibody described below), One skilled in the art will appreciate that the expression can be used. Control antibody preparations (eg, purified immunoglobulins from the same animal species prior to immunization) can be isotype-matched and species-matched antibodies of the invention. Those skilled in the art will recognize tests with control antibodies to demonstrate appropriate and critical specificity.

  In some embodiments of the invention, an antibody that specifically binds to a target molecule is at least 5-, 10-, or 20-fold higher than the detection signal emitted by other proteins when used in an immunochemical assay. A detection signal is emitted. In some embodiments, an antibody that specifically binds to the target molecule does not detect other proteins in an immunochemical assay and can immunoprecipitate the target molecule from solution.

In some embodiments, the immunoglobulin chain may comprise a variable region and a constant region in the 5 ′ to 3 ′ direction. The variable region may comprise the structure of three complementarity determining regions (CDRs) interspersed with framework (FR) regions, ie FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The invention also encompasses heavy or light chain variable regions, framework regions, and CDRs. An antibody of the invention may comprise a heavy chain constant region comprising part or all of the CH1 region, hinge, CH2 and CH3 regions. An antibody of the invention may comprise a light chain constant region comprising part or all of a CL region. Antibodies of the present invention can have the following K _D values 1 × 10 ^-7 M for the target molecule. In other embodiments, the antibody, the target molecule and _the K _D value ^{1 × 10 -8 M, 1 ×} 10 -9 M, 1 × 10 -10 M, 1 × 10 -11 M, 1 × 10 -12 M or Join below that. In certain embodiments, K _D value is 1pM~500pM, 500pM~1μM, 1μM~100nM or 100mM~10nM,.

  The antibody of the present invention may be derived from any animal species such as mammals. Exemplary natural antibodies include, but are not limited to, genetically engineered rodents produced by genetic engineering to produce human, camelid (eg, camels and llamas), chicken, goat, and human antibodies. Antibodies from rodents (e.g., rats, mice, hamsters and rabbits), including Lonberg et al., WO 93/12227, which is hereby incorporated by reference in its entirety. U.S. Pat. No. 5,545,806; and Kucherlapati et al., WO 91/10741; U.S. Pat. No. 6,150,584). A natural antibody is an antibody produced by a host animal. “Gene engineered antibody” refers to an antibody having an amino acid sequence different from that of the original antibody. Since the recombinant DNA technology in this application is valid, it need not be limited to the amino acid sequences found in natural antibodies, and the antibodies can be redesigned to obtain the desired characteristics. The possible variations are many, ranging from changing one or a few amino acids to complete redesigns (eg variable or constant regions). Changes in the constant region are usually made to improve or modify features such as complement fixation, membrane interaction, and other effector functions. Changes in the variable region are made to improve antigen binding characteristics.

  Other antibodies are specifically intended to be oligoclonal antibodies. As used herein, the phrase “oligoclonal antibody” refers to a predetermined mixture of different monoclonal antibodies. See, for example, PCT International Publication No. 95/20401; US Pat. Nos. 5,789,208 and 6,335,163. In one embodiment, an oligoclonal antibody consisting of a predetermined mixture of antibodies to one or more epitopes is produced in a single cell. In other embodiments, the oligoclonal antibody comprises a plurality of heavy chains that can pair with a common light chain to produce an antibody with multispecificity (eg, PCT Publication No. WO 04/009618). Oligoclonal antibodies are particularly useful when it is desirable to target multiple epitopes on a single target molecule. In view of the assays and epitopes disclosed herein, one of ordinary skill in the art can make or select an antibody or mixture of antibodies that can be adapted for its intended purpose and desired needs.

  The present invention also includes the recombinant antibody of the present invention. In the present application, these recombinant antibodies have the same amino acid sequence as the natural antibody or have an amino acid sequence modified from the natural antibody. The recombinant antibodies can be generated in any expression system, including both prokaryotic and eukaryotic expression systems, or using phage display methods (e.g., incorporated herein by reference in their entirety). Dower et al., WO 91/17271 and McCafferty et al. WO 92/01047; U.S. Pat.No. 5,969,108, U.S. Pat.No. 6,331,415; U.S. Pat. Wanna).

Antibodies can be designed in a number of ways. They can be produced as single chain antibodies (such as low modular immunopharmaceuticals or SMIP ™), Fab and F (ab ′) ₂ fragments, and the like. The antibody can be a humanized antibody, a chimeric antibody, a deimmunized antibody, or a fully human antibody. Many antibody types and methods for designing such antibodies are described in numerous publications. See, e.g., U.S. Patent Nos. 6,355,245; 6,180,370; 5,693,762; 6,407,213; 6,548,640; 5,565,332; 5,225,539; I want to be.

  The genetically modified antibody should be functionally equivalent to the natural antibody described above. In certain embodiments, the modified antibody provides improved stability or / and therapeutic effect. Examples of modified antibodies include those involving conservative substitution of amino acid residues and one or more deletions or additions of amino acids that do not significantly detrimentally alter antigen binding utility. Substitutions can range from changing or modifying one or more amino acid residues to complete redesign of a region as long as therapeutic utility is maintained. The antibody of the present invention can be modified after translation (for example, acetylation and / or phosphorylation) or can be modified synthetically (for example, attachment of a labeling group).

  Antibodies with engineered or mutated constant or Fc regions are useful for modulating effector functions such as, for example, antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) obtain.

  In certain embodiments, genetically modified antibodies are chimeric antibodies and humanized antibodies.

  A chimeric antibody is an antibody having portions derived from different antibodies. For example, a chimeric antibody can have variable and constant regions derived from two different antibodies. Donor antibodies can be derived from different species. In certain embodiments, the variable region of the chimeric antibody is non-human (eg, mouse) and the constant region is human.

  Genetically modified antibodies used in the present invention include humanized antibodies grafted with CDRs. In one embodiment, the humanized antibody has a heavy and / or light chain CDR of a non-human donor immunoglobulin and a heavy and light chain framework and constant region of a human acceptor immunoglobulin. Methods for making humanized antibodies are disclosed in U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; Incorporated in the book.

In some embodiments, an antibody of the invention comprises a human immunoglobulin that has all or substantially all of the framework regions in which one or more CDR regions are present as a synthetic amino acid sequence that specifically binds to a target molecule. It contains at least one, typically substantially all two of the variable regions (Fab, Fab ′, F (ab ′) ₂ , Fabc, Fv, etc.) that are the framework regions of the consensus sequence. The framework region may be the original human immunoglobulin sequence. CDR regions in other antibodies can be selected to have human immunoglobulin consensus sequences in such CDRs or sequences of the original human antibody. Optimally, the antibody also comprises at least a portion of an immunoglobulin constant region (Fc) of a human immunoglobulin. Ordinarily, the antibody will contain both the light chain as well as at least the variable region of a heavy chain. The antibody can also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain.

  Methods for identifying antibody CDR regions by amino acid sequence analysis of antibodies are well known (e.g., Wu, TT and Kabat, EA (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203: 121-53 (1991); Morea et al., Biophys Chem. 68 (1-3): 9-16 (Oct. 1997); Morea et al., J Mol Biol. 275 (2): 269-94 (Jan .1998); Chothia et al., Nature 342 (6252): 877-83 (Dec. 1989); see Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007)).

  By way of example and not limitation, the following method can be used to identify antibody CDRs.

  In the case of CDR-L1, CDR-L1 is about 10-17 amino acid residues in length. In general, start at the residue at about position 24 (the residue before the residue at position 24 is typically a cysteine). CDR-L1 ends at the residue before the tryptophan residue. Typically, the tryptophan-containing sequence is one of Trp-Tyr-Gln, Trp-Leu-Gln, Trp-Phe-Gln, or Trp-Tyr-Leu, and is the last residue in the CDR-L1 region. The group is the residue before TRP in all these sequences.

  In the case of CDR-L2, CDR-L2 is typically 7 residues in length. Normally, CDR-L2 starts about 16 residues after the CDR-L1 terminus, and typically starts at the residue after the Ile-Tyr, Val-Tyr, Ile-Lys, or Ile-Phe sequence.

  In the case of CDR-L3, CDR-L3 is typically 7-11 amino acid residues in length. Usually, this region starts about 33 residues after the end of the CDR-L2 region. The residue before the region start is often cysteine, ending with the residue before Phe in the sequence Phe-Gly-XXX-Gly (where XXX is the three letter representation of any single amino acid).

  In the case of CDR-H1, the CDR-H1 region is typically 10-12 amino acid residues in length, often starting from the residue at about position 26. The region typically starts 4 or 5 residues after the cysteine residue, typically Trp (Trp is often one of the following sequences: Trp-Val, Trp-Ile, or Trp-Ala. Ends with the residue before (found in one). In the case of CDR-H2, the CDR-H2 region is typically 16-19 residues in length, and typically begins 15 residues after the end residues of the CDR-H1 region. The region is typically of the sequence Lys / Arg-Leu / Ile / Val / Phe / Thr / Ala-Thr / Ser / Ile / Ala (such as the sequences Lys-Leu-Thr and Arg-Ala-Ala). End with the previous amino acid residue.

  In the case of CDR-H3, the CDR-H3 region is typically 3-25 amino acids in length, typically 33 amino acid residues after the terminal residues of the CDR-H2 region (often, cysteine residues For example, starting from 2 amino acid residues after cysteine in the sequence Cys-Ala-Arg). The region ends with the amino acid immediately preceding Trp in the sequence Trp-Gly-XXX-Gly (where XXX is a three letter representation of any single amino acid).

In one embodiment of the present application, the antibody fragment is a truncated chain (carboxyl terminal truncated). In certain embodiments, these truncated chains have one or more immunoglobulin activities (eg, complement fixation activity). Examples of truncated chains include Fab fragments (consisting of VL, VH, CL and CH1 regions); Fd fragments (consisting of VH and CH1 regions); Fv fragments (consisting of single-chain VL and VH regions of the antibody) dAb fragments (consisting of VH regions); isolated CDR regions; (Fab ') ₂ fragments; bivalent fragments (including 2 Fab fragments linked by hinge region disulfide bonds) It is not limited to. Each truncated strand can be produced by conventional biochemical techniques (such as enzymatic cleavage) or recombinant DNA techniques known in the art. These polypeptide fragments can be obtained by proteolytic cleavage of intact antibodies by methods well known in the art, or by site-directed (after CH1 to produce Fab fragments, or the hinge region to produce (Fab ') ₂ fragments). Can be produced by inserting a stop codon at the desired position in the vector using mutagenesis (such as later). Single chain antibodies can be produced by linking DNA encoding peptide linkers linked to VL and VH protein fragments to the VL and VH coding regions.

“Fv” usually refers to the minimum antibody fragment which contains a complete antigen recognition and binding site. This region is composed of a dimer in which one heavy chain variable region and one light chain variable region (ie, VL region and VH region) are tightly and non-covalently bound. In this configuration, the three CDRs of each variable region interact to form an antigen binding site profile on the surface of the V _H -V _L dimer. In summary, CDRs provide antigen binding specificity for antibodies. However, even one variable region (or half of an Fv containing three antigen-specific CDRs) has the ability to recognize and bind antigen, although perhaps with less affinity than the complete binding site. “Single-chain Fv” or “scFv” antibody fragments comprise the V _H and V _L regions of an antibody, wherein these regions are present in a single polypeptide chain. In certain embodiments, Fv polypeptide further, scFv is it possible to form the desired structure for antigen binding, including a polypeptide linker between the V _H and V _L regions. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, edited by Rosenburg and Moore (Springer-Verlag: New York, 1994), pp. 269-315.

Papain digestion of intact antibodies results in two identical antigen-binding fragments, each called a “Fab” fragment, each with a single antigen-binding site, and the remaining “ Fc "fragments are produced. The Fab fragment contains the complete light chain (ie, the light chain constant region (CL) and variable region (VL)), as well as the first constant region (CH1) and variable region (VH) of the heavy chain. Fab ′ fragments differ from Fab fragments in that several residues are added to the carboxy terminus of the heavy chain CH1 region containing one or more cysteines derived from the antibody hinge region. Fab′-SH herein is the name for Fab ′ in which the cysteine residue (s) in the constant region have a free thiol group. F (ab ′) ₂ antibody fragments were originally produced as pairs of Fab ′ fragments which have hinge cysteines between them. For example, pepsin treatment of antibodies can still antigen cross-link while providing an F (ab ′) ₂ fragment with _two antigen binding sites. That is, the F (ab ′) ₂ fragment contains two disulfides that bind to the Fab fragment. Other chemical couplings of antibody fragments are also known. Thus, in certain embodiments, an antibody of the invention recognizes an E746-A750 deletion in EGFR or specifically binds to an E746-A750 deletion or recognizes an L858R point mutation or an L858R point mutation. It may contain 1, 2, 3, 4, 5, 6, or more specifically binding CDRs. In some embodiments, an antibody of the invention comprises at least one complementarity determining region (CDR), wherein the CDR is SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 17, and It specifically binds to an EGFR E746-A750 deletion type comprising a sequence selected from the group consisting of SEQ ID NO: 18. In some embodiments, an antibody of the invention that specifically binds to an EGFR L858R mutation comprises at least one complementarity determining region (CDR), wherein the CDR is SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25. An amino acid sequence selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32.

  Another antibody type of the present invention is SMIP. SMIP is a type of single-chain peptide designed to include an antigen binding region and an effector region (CH2 and CH3 regions). See, for example, US Patent Application 20050238646. The antigen binding region can be derived from the variable region or CDR of an antibody (eg, an EGFR L858R point mutation specific antibody of the invention). Alternatively, the antigen is derived from a protein that specifically binds to a designated target (eg, a non-immunoglobulin molecule that binds to an EGFR L858R mutant molecule).

  Bispecific antibodies can be monoclonal, human or humanized antibodies that have binding specificities for at least two different antigens. As used herein, one of the binding specificities is a target molecule of the invention (e.g. EGFR L858R mutant or EGFR E746-A750del mutant), the other is e.g. a cell surface protein or receptor or receptor To any other antigen, such as a body subunit. Alternatively, the therapeutic agent can be placed on the chain (eg, heavy chain) of the antibody. The therapeutic agent can be a drug, toxin, enzyme, DNA, radionuclide, and the like.

In some embodiments, the antigen-binding fragment can be a diabody. The term `` diabody '' refers to a small antibody fragment having two antigen-binding sites that are linked to the light chain variable region (V _L ) in the same polypeptide chain (V _H -V _L ). Contains the heavy chain variable region (V _H ). The fragment is paired between chains rather than within the V region, resulting in a multivalent fragment (i.e., a fragment with two antigen binding sites), resulting in a VH and VL region. Can be prepared by constructing scFv fragments with short linkers (about 5-10 residues). Because the linker is too short to pair between two regions on the same chain, the region necessarily pairs with the complementary region of the other chain, creating two antigen binding sites. Diabodies are described in detail, for example, in EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

  Camelid antibodies refer to a unique antibody type lacking a light chain, initially found in camelid family animals. These heavy chains, called so-called heavy chain antibodies, bind the antigen in one single region, the variable region of an immunoglobulin heavy chain called VHH. VHH shows homology with the heavy chain variable region of the human VHIII family. VHH from immunized camels, dromedaries, or llamas has several advantages such as efficient production in microorganisms such as Saccharomyces cerevisiae. In certain embodiments, single chain antibodies, and chimeric, humanized or primatized (CDR grafted) antibodies, and chimeric single chain antibodies or CDR grafted single chain antibodies comprise a heterologous portion. And is encompassed by the present disclosure as an antigen-binding fragment of an antibody. The various portions of these antibodies can be chemically coupled by conventional techniques or can be prepared as a continuous protein using genetic engineering techniques. For example, a nucleic acid encoding a chimeric or humanized chain can be expressed to produce a continuous protein. For example, U.S. Pat.Nos. 4,816,567 and 6,331,415; U.S. Pat. See 0,239,400 B1. See also Newman et al., BioTechnology, 10: 1455-1460 (1992) for primatized antibodies. See, for example, Ladner et al., US Pat. No. 4,946,778; and Bird et al., Science, 242: 423-426 (1988) for single chain antibodies.

  In addition, functional fragments of antibodies, including fragments of chimeric, humanized, primatized or single chain antibodies, can also be produced. A functional fragment of an antibody of the invention retains at least one binding and / or regulatory function of the full-length antibody from which the functional fragment is derived. Since immunoglobulin-related genes contain separate functional regions, each with one or more different biological activities, antibody fragment genes may be related to other genes (e.g., enzymes, U.S. patents incorporated by reference in their entirety) No. 5,004,692) can be fused with a functional region to produce a fusion protein or conjugate having novel properties.

  Non-immunoglobulin binding polypeptides are also contemplated. To make a non-immunoglobulin binding agent, for example, a CDR from the antibody disclosed herein can be inserted into a suitable non-immunoglobulin scaffold. Suitable scaffold construct candidates can be derived, for example, from fibronectin type III and cadherin superfamily members.

  In addition, other equivalent non-antibody molecules are contemplated, such as protein binding regions or aptamers that specifically bind to a target molecule described herein (eg, an EGFR mutant). See, for example, Neuberger et al., Nature 312: 604 (1984). Aptamers are oligonucleic acid or peptide molecules that bind to specific target molecules. DNA or RNA aptamers are typically short oligonucleotides made by repeating rounds of selection to bind to a target molecule. Peptide aptamers typically consist of variable peptide loops that bind to both ends of the protein scaffold. This dual structural limitation usually increases the peptide aptamer binding affinity to antibody equivalents (nanomolar range).

  The present invention also discloses the use of antibodies having immunotoxins. Conjugates that are immunotoxins such as antibodies have been widely described in the art. The toxin can be coupled to the antibody by conventional coupling techniques, or an immunotoxin containing a protein toxin moiety can be produced as a fusion protein. In certain embodiments, the antibody conjugate may include a stable linker and release a cytotoxic agent into the cell (see US Pat. Nos. 6,867,007 and 6,884,869). The conjugate of the present application can be used in a method corresponding to the acquisition of such an immunotoxin. Examples of such immunotoxins are described in Byers et al., Seminars Cell Biol 2: 59-70 (1991) and Fanger et al., Immunol Today 12: 51-54 (1991). Exemplary immunotoxins include radiotherapy drugs, ribosome inactivating protein (RIP), chemotherapeutic drugs, toxic peptides, or toxic proteins.

  The specific antibodies disclosed in the present invention may be used alone or in combination. Antibodies may be used in large quantities in array form. Antibody microarrays are a collection of immobilized antibodies that are typically spotted and immobilized on a solid surface (such as glass, plastic and silicon chips).

  In certain embodiments, the antibodies disclosed in the present invention are specifically designated for diagnostic and therapeutic applications, as described herein. Thus, antibodies can be used for treatment (including combination therapy), disease diagnosis and prognosis, and monitoring disease progression. Accordingly, the present invention further includes compositions comprising an antibody or antigen-binding portion of one or more embodiments of the present invention as described herein. The composition may further comprise a pharmaceutically acceptable carrier. The composition comprises two or more antibodies or antigen binding portions each having specificity for different target sites of the invention, or two or more different antibodies or antigen binding portions that are specific for the same site of the invention Can be included. The composition of the present invention may comprise one or more antibodies or antigen-binding portions of the present invention and one or more additional reagents, diagnostics or therapeutics.

  The present application provides polynucleotide molecules encoding the antibodies and antibody fragments and analogs thereof described herein. Due to the degeneracy of the genetic code, various nucleic acid sequences encode each antibody amino acid sequence. The desired nucleic acid sequence can be produced by novel solid phase DNA synthesis or by PCR mutagenesis of the desired polynucleotide variant initially prepared. In one embodiment, the codons used include those typical for humans or mice (see, eg, Nakamura, Y., Nucleic Acids Res. 28: 292 (2000)).

  The binding agent of the present invention includes an antibody having the amino acid sequence described herein (whether or not the leader sequence is included), and the binding agent includes one or more CDR regions (heavy chain) of the present invention. Or at least 6 contiguous amino acids containing the amino acid sequence (from either or both of the light chains) and at least 90%, or at least 95%, or at least 96%, 97% of the polypeptide. 98% or 99% identical (e.g., SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 30, 90% identical, or at least 95% identical, or at least 96%, 97%, 98% or 99% identical) to SEQ ID NO: 31 or SEQ ID NO: 32.

  “Identical percentage (%)” of two polypeptides or two polynucleotides (or “percent identity”) refers to the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix (registered trademark), Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711) and default settings for similarity determination, comparing the amino acid sequences of two polypeptides or comparing the nucleotide sequences of two polynucleotides Intended for the resulting similarity score. Bestfit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2: 482-489 (1981)) to find the best segment of similarity between two sequences.

  As an example, but not limited thereto, a polypeptide having the amino acid sequence that is at least 95% identical to the reference amino acid sequence of the polypeptide binding agent of the present invention is, for example, that the polypeptide sequence is each of the reference amino acid sequences. It is intended that the amino acid sequence of the polypeptide is identical to the reference sequence, except that it may contain up to 5 amino acid modifications per 100 amino acids. That is, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the reference amino acid sequence, up to 5% of the amino acid residues of the reference sequence can be deleted or replaced with another amino acid, or Some amino acids of up to 5% of the total amino acid residues can be inserted into the reference sequence. These reference sequence modifications can occur at the amino-terminal or carboxy-terminal position of the reference amino acid sequence or any position between the terminal positions, between residues in the reference sequence (individually), or one or more in the reference sequence It can be interspersed in any of a number of consecutive groups.

  Similarly, a reference nucleotide sequence that encodes a binding agent of the invention and at least a polynucleotide having, for example, 95% “identical” nucleotide sequence, the polypeptide sequence encodes a binding agent or antibody of the invention It means that the nucleotide sequence of the polypeptide is identical to the reference sequence, except that it may contain up to 5 point mutations for each 100 nucleotides of the reference nucleotide sequence. For example, to obtain a polypeptide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or replaced with another nucleotide, or the total of the reference sequence Some nucleotides of up to 5% of the nucleotides can be inserted into the reference sequence. These mutations in the reference sequence can occur at the 5 terminal position of the reference nucleotide sequence or any position between the end positions, between the nucleotides of the reference sequence (individually), or one or more consecutive in the reference sequence Can be interspersed with any of the groups.

  When using a Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence of the invention, it will be appreciated that the reference amino acid sequence or reference nucleotide As a percentage of identity is calculated over the full length of the sequence, and there is a gap in homology of up to 5% of the total number of amino acid residues (in the polypeptide) or nucleotide residues (in the polynucleotide) in the reference sequence. Parameters are set as allowed.

  In a further aspect of the invention, a binding agent that specifically binds to an epidermal growth factor receptor (EGFR) molecule having a point mutation in which leucine at position 858 is replaced by arginine or an epithelium that is defective at position E746-A750 A polynucleotide encoding a binding agent that specifically binds to a growth factor receptor (EGFR) molecule is provided.

The terms “polynucleotide”, “nucleic acid molecule”, and “nucleic acid sequence” are used interchangeably herein and include, but are not limited to, DNA, RNA, DNA / RNA hybrids, modifications thereof, and the like. Refers to a polymer of nucleotides of any length that is not. Unless otherwise specified, nucleotides are written in the 5 'to 3' direction when presenting nucleotide sequences. Thus, nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. Modifications to the nucleotide structure, if present, can be given before or after polymer assembly. The nucleotide sequence can be interrupted by non-nucleotide components. The polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, “caps”, substitution with one or more analogs of natural nucleotides, such as uncharged linkages (eg, methylphosphonates, phosphotriesters, phosphoamidos). Date, carbamate, etc.) and internucleotide modifications such as modifications with charged linkages (e.g. phosphorothioate, phosphorodithioate, etc.), e.g. proteins (e.g. nucleases, toxins, antibodies, signal peptides, poly-L-lysine etc. Modifications containing pendant moieties such as, modifications with intercalators (e.g., acridine, psoralen, etc.), modifications containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), modifications containing alkylating agents Modification by a modified linkage (e.g. Non-modified forms of the nucleotide (s) are included. Furthermore, whether any hydroxyl group normally present in the sugar is replaced by, for example, a phosphonate group, a phosphate group, protected with a standard protecting group, or activated to prepare additional linkages to additional nucleotides. Or can be bound to a solid support. The 5 ′ and 3 ′ terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups. Polynucleotides include similar forms of ribose or deoxyribose sugars commonly known in the art, such as 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'- Azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugars, acetoheptulose, acyclic analogs and abasic nucleoside analogs such as methylriboside And so on. One or more phosphodiester linkages can be replaced by alternative linking groups. These alternative linking groups include phosphates such as P (O) S (`` thioate ''), P (S) S (`` dithioate ''), `` (O) NR <sub>2</sub> (`` amidate ''), P (O) R, P (O) OR ′, CO or CH ₂ (“form acetal”), wherein each R or R ′ is independently H or optionally substituted or unsubstituted alkyl containing an ether (—O—) linkage ( 1-20C), aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl) is included, but is not limited to. Not all linkages in a polynucleotide need be the same. The above description applies to all polynucleotides referred to herein (including RNA and DNA).

  The present application also provides polynucleotide molecules encoding analogs of the binding agents (eg, antibodies) described herein. Due to the degeneracy of the genetic code, several different nucleic acid sequences can encode each antibody amino acid sequence. The desired nucleic acid sequence can be produced by novel solid phase DNA synthesis or by PCR mutagenesis of the desired polynucleotide variant initially prepared. In one embodiment, the codons used include those typical for humans, rabbits, or mice (see, eg, Nakamura, Y., Nucleic Acids Res. 28: 292 (2000)).

  Furthermore, the present invention provides, in part, for producing an isolated polynucleotide encoding a binding agent of the invention, a nucleotide probe that hybridizes such a polynucleotide, and a recombinant fusion polypeptide. Methods of using such polynucleotides, vectors, and host cells are provided. Unless otherwise specified, all nucleotide sequences determined by DNA molecular sequencing herein are determined using an automated DNA sequencer (such as Model 373 from Applied Biosystems, Inc.) and are described herein. All amino acid sequences of the polypeptides encoded by the DNA molecules determined in 1 were determined using an automated peptide sequencer. As is known in the art, in any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. The nucleotide sequence determined by automated manipulation is typically at least about 90% identical to the actual nucleotide sequence of the sequenced DNA molecule, more typically at least about 95% to about 99.9%. Are the same. The actual sequence can be more accurately determined by other approaches such as manual DNA sequencing methods well known in the art. Also, as is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence indicates that the deduced amino acid sequence encoded by the determined nucleotide sequence is the sequence It causes a frameshift in the translation of the nucleotide sequence starting from such an insertion or deletion position so that it is completely different from the amino acid sequence actually encoded by the determined DNA molecule. Unless otherwise specified, each nucleotide sequence described herein is represented as a sequence of deoxyribonucleotides (abbreviated A, G, C, and T). However, the “nucleotide sequence” of a nucleic acid molecule or polynucleotide refers to a deoxyribonucleotide sequence with respect to a DNA molecule or polynucleotide, and with respect to an RNA molecule or polynucleotide, the corresponding sequence of ribonucleotides (A, G, C And U), where each thymidine deoxyribonucleotide (T) in the designated deoxyribonucleotide sequence is replaced by a ribonucleotide uridine (U). For example, the notation of an RNA molecule having the sequence of SEQ ID NO: 1 or described using the abbreviations for deoxyribonucleotides is that each deoxyribonucleotide A, G or C of SEQ ID NO: 1 corresponds to the corresponding ribonucleotide A, Or is intended to indicate an RNA molecule having a sequence that is substituted by C and in which each deoxyribonucleotide T is replaced by ribonucleotide U.

  In some embodiments of the invention, an isolated polynucleotide comprising (or complementary to) a nucleotide sequence that is at least about 95% identical to a sequence comprising SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7. Isolated polynucleotide). In some embodiments of the invention, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, A nucleotide sequence that is at least about 95% identical to a nucleotide sequence encoding an antibody (or fragment thereof) comprising the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32. An isolated polynucleotide comprising (or an isolated polynucleotide complementary thereto) is provided.

  Using the information provided herein (such as the nucleotide sequence specified in SEQ ID NO: 1, 3, 5, or 7), using standard cloning (such as cDNA cloning) and screening procedures using mRNA as the starting material Thus, the nucleic acid molecule of the present invention encoding the polypeptide binding agent (for example, antibody) of the present invention can be obtained.

  As described, the present invention provides, in part, full-length antibodies. According to the signal hypothesis, proteins secreted by mammalian cells have a signal or secretory leader sequence that is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum is initiated. Most mammalian cells, and even insect cells, cleave secreted proteins with the same specificity. However, in some cases, cleavage of the secreted protein is not completely uniform, which results in two or more mature species on the protein. Furthermore, the cleavage specificity of the secreted protein is ultimately determined by the primary structure of the complete protein, ie, the cleavage specificity of the secreted protein is intrinsic to the amino acid sequence of the polypeptide. It has been known for a long time. Thus, the present invention provides, in part, SEQ ID NO: 1, 3, 5, or 7 having an additional nucleic acid residue located at the 5 ′ to 5 ′ terminal residue of SEQ ID NO: 1, 3, 5, or 7. Encodes an intact antibody having the nucleotide sequence defined in 7 (e.g., containing 2 heavy chains and 2 light chains), and the N-terminal residue from SEQ ID NO: 2, 4, 6 or 8 Nucleotide sequences that encode the amino acid sequence of an intact antibody chain having the amino acid sequence defined in SEQ ID NO: 2, 4, 6, or 8 with an additional amino acid residue located in the group are provided. Similarly, the present invention includes CDRs of the present invention (e.g., SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, A CDR that requires an additional nucleic acid residue located at the 5 ′ to 5 ′ terminal residue of a polynucleotide that encodes a CDR comprising the amino acid sequence defined in SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32) An encoding nucleotide sequence is also provided.

  In some embodiments, the polynucleotide encoding the antibody or binding agent comprises a nucleotide sequence as defined in SEQ ID NO: 1, 3, 5, or 7. In some embodiments, the polynucleotide encoding an antibody or binding agent comprises SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 23, sequence A nucleotide sequence encoding a CDR having the amino acid sequence set forth in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32; In some embodiments, the polynucleotide encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, or 8.

  As described, the polynucleotides of the invention may be in the form of RNA, such as mRNA, or in the form of DNA, such as cDNA and genomic DNA obtained, for example, by cloning or synthetically produced. DNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be the coding strand (also known as the sense strand) or the non-coding strand (also called the antisense strand).

  An isolated polynucleotide of the present invention can be a nucleic acid molecule (DNA or RNA) that has been removed from the native environment of the polynucleotide. For example, a vector-containing recombinant DNA molecule is considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or (partially or substantially) purified DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Furthermore, isolated nucleic acid molecules of the present invention include such molecules produced synthetically.

  The polynucleotide of the present invention comprises a nucleic acid molecule having a sequence defined by SEQ ID NOs: 1, 3, 5, and 7, a nucleic acid molecule comprising a coding sequence for an antibody, and a sequence different from the above but a reduced gene code. A binding agent of the invention which encodes an antibody or binding agent of the invention by weight. Since the genetic code is well known in the art, it will be routine for those skilled in the art to make such degenerate variants.

  The invention further provides an isolated polynucleotide comprising a nucleotide sequence having a sequence that is complementary to one of the polynucleotides encoding the binding agent of the invention or encoding the antibody of the invention. Such isolated molecules, especially DNA molecules, are used as probes for genetic mapping by in situ hybridization of chromosomes, as well as for detection of antibody expression in tissues (e.g., human tissues), e.g., by Northern blot analysis. Useful.

  In some embodiments, the binding agents (eg, antibodies) of the invention are encoded by at least a portion of the nucleotide sequences described herein. As used herein, a “part” or “fragment” is a sequence fragment comprising several consecutive amino acid residues (in the case of a polypeptide fragment (sometimes referred to herein as a peptide)) Means a sequence fragment (e.g. a 50 nucleotide sequence is part of a 100 nucleotide sequence in length) containing several nucleotide residues (in the case of polynucleotide fragments) less than such residues in the complete sequence . That is, the fragment of the specified molecule is smaller than the specified molecule. For example, a polynucleotide encoding a binding agent of the invention and / or a polynucleotide encoding an antibody may comprise a portion of an intron sequence that does not encode any amino acid in the generated binding agent or antibody. Polynucleotide fragments can be at least about 15 nucleotides, or at least about 20 nucleotides, or at least about 30 nucleotides, or at least about 40 nucleotides in length and are useful as diagnostic probes and primers as discussed herein. . Of course, in the case of larger fragments of about 50-1500 nucleotides in length, the fragments are mostly if not all of the antibody or binding agent encoding the nucleotide sequence of the cDNA having the sequences described herein. Therefore, it is still useful in the present invention. The present invention provides a polynucleotide (eg, a purified polynucleotide) encoding a binding agent that specifically binds to an epidermal growth factor receptor (EGFR) molecule that is deficient at position E746-A750. For example, “a fragment of at least 20 nucleotides in length” means a fragment comprising 20 or more consecutive nucleotides in each nucleotide sequence derived from the fragment.

  Polynucleotide fragments are described, for example, in Molecular Cloning, A Laboratory Manual, 2nd. Edition, Sambrook, J., Fritsch, EF and Maniatis, T., Ed., Cold Spring Harbor Laboratory In the diagnostic use of nucleotide probes according to conventional DNA hybridization techniques as described in Press, Cold Spring Harbor, NY (1989), or as primers for amplification of target sequences by polymerase chain reaction (PCR) Useful in the use of It will be appreciated that polynucleotides that hybridize only with a poly A sequence or only with a complementary stretch of T (or U) residues may be used to hybridize with portions of the nucleic acids of the invention. Is not included in the polynucleotide. This is because such polynucleotides hybridize to any nucleic acid molecule (eg, virtually any double-stranded cDNA clone) that contains a poly (A) extension or its complement. Generation of such DNA fragments is routine for those skilled in the art, and by the examples described herein, by restriction endonuclease cleavage or shearing, by sonication of DNA available from cDNA clones, or as described herein. Can be accomplished by synthesis with the sequences disclosed in the literature. Alternatively, such fragments can be made directly synthetically.

In another aspect of the invention, there are provided isolated polynucleotides (eg, nucleotide probes) that hybridize under stringent conditions with a polynucleotide encoding a binding agent of the invention or encoding an antibody. The term `` stringent conditions '' for nucleotide sequence or nucleotide probe hybridization conditions is from about T _m -5 ° C. (ie, 5 ° C. below the melting temperature (T _m ) of the probe or sequence) to about 20 ° C. below T _m. “Stringency” that occurs within a low range of ˜25 ° C. Typical stringent conditions are 50% formamide, 5 × SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, and 20 mg / Overnight incubation at 42 ° C. in a solution containing mL denatured sheared salmon sperm DNA followed by filter wash in 0.1 × SSC at about 65 ° C. As will be appreciated by those skilled in the art, the stringency of hybridization can be altered to identify or detect identical or related polynucleotide sequences.

  A polynucleotide or nucleotide probe that hybridizes with a reference polynucleotide is hybridized with a full-length reference polynucleotide, or at least about 15 nucleotides (nt), or at least about 20 nt, or at least about 30 nt of the reference polynucleotide, or A polynucleotide or nucleotide probe (eg, a DNA, RNA, or DNA-RNA hybrid) that hybridizes with a portion of a reference polynucleotide that is about 30-70 nt is contemplated. These nucleotide probes of the invention are useful as diagnostic probes and primers (eg, for PCR) as discussed herein.

  Of course, a polynucleotide that hybridizes to a larger portion of a reference polynucleotide, e.g., a 50-750 nt portion of the reference polynucleotide, or even a full-length reference polynucleotide, is the nucleotide of the cDNA described herein. Since it corresponds to most if not all of the sequences or nucleotide sequences defined in SEQ ID NOs: 1, 3, 5, and 7, it is useful as a probe of the present invention.

  As described, a nucleic acid molecule of the invention encodes a binding agent of the invention and includes, but is not limited to, the amino acid sequence of an intact mature polypeptide itself; a fragment thereof; a coding for a mature polypeptide Coding sequences for sequences and additional sequences (e.g. sequences encoding leader or secretory sequences such as pre-, or pro- or pre-pro-protein sequences); with or without said additional coding sequences Additional non-coding sequences (e.g., transcribed and untranslated sequences that play a role in mRNA processing such as transcription and splicing, and polyadenylation signals, e.g., for ribosome binding and stability of mRNA) Including introns and non-coding 5 'and 3' sequences Having limiting), the mature coding sequence of the polypeptide; comprising additional amino acids (additional coding sequence which encodes the like) that provide additional functionality, the.

  Thus, a sequence encoding a polypeptide can be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused polypeptide. In a particular embodiment of this aspect of the invention, the marker amino acid sequence is a hexahistidine peptide, such as a tag provided in a pQE vector (Qiagen, Inc.), many of which are commercially available. . For example, hexahistidine, as described in Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824 (1989), provides convenient purification of the fusion protein. The “HA” tag is another peptide useful for purification corresponding to an epitope derived from the influenza hemagglutinin protein described in Wilson et al., Cell 37: 767 (1984). Other such fusion proteins as discussed below include the binding agents and / or antibodies of the invention fused to the Fc region at the N-terminus or C-terminus.

  The invention further relates to variants of the nucleic acid molecules of the invention that encode portions, analogs or derivatives of the binding agents or antibodies disclosed herein. Like a natural allelic variant, a variant can exist in nature. By “allelic variant” is intended one of several alternative forms of a gene occupying a given locus on the chromosome of an organism. See, for example, Genes II, Lewin, B., John Wiley & Sons, New York (1985). Non-naturally occurring variants can be produced using mutagenesis techniques known in the art.

  Such variants include variants produced by nucleotide substitutions, deletions or additions. Substitutions, deletions or additions can include one or more nucleotides. Variants can be modified in the coding region, non-coding region, or both. Modifications in the coding region can result in conservative or non-conservative amino acid substitutions, deletions or additions. Some modifications encompassed by the present invention are silent substitutions, additions and deletions that do not alter the properties and activities (eg, specific binding activities) of the binding agents and / or antibodies disclosed herein.

  A further embodiment of the invention encompasses an isolated polynucleotide comprising a nucleotide sequence that is at least 90% identical. In some embodiments of the invention, the nucleotide is at least 95%, 96%, 97%, 98% or 99% identical to a polynucleotide encoding a binding agent of the invention or encoding an antibody.

  In practice, for example, any particular nucleic acid molecule is at least against the nucleotide sequence defined in SEQ ID NOs: 1, 3, 5, and 7 or the nucleotide sequence of a cDNA clone encoding a CDR described herein Whether it is 90%, 95%, 96%, 97%, 98% or 99% identical is determined by the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix®, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711) can be used for conventional judgment.

  Due to the degeneracy of the genetic code, the nucleic acid sequence of the cDNA described herein, the nucleic acid sequence defined in SEQ ID NO: 1, 3, 5, or 7, or SEQ ID NO: 2, 4, 6, 8, 9, 10, A nucleic acid sequence encoding an amino acid sequence defined in 11, 16, 17, 18, 23, 24, 25, 30, 31, or 32 and at least 90%, 95%, 96%, 97%, 98%, or 99 Those of skill in the art will readily recognize that a large number of nucleic acid molecules having the same sequence encode a polypeptide having specific binding activity. In fact, since degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be apparent to those skilled in the art without the need for the above comparison assay. It will be further recognized in the art that for such nucleic acid molecules that are not degenerate variants, a reasonable number also encodes a polypeptide that retains the specific binding activity of a reference binding agent or antibody of the invention. I will. This is because those skilled in the art are well aware of amino acid substitutions that are unlikely or unlikely to cause significant protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid). is there. For example, guidance on how to create phenotypically silent amino acid substitutions can be found in Bowie et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” Science 247: 1306-1310 (1990). Two main approaches for studying the tolerance of amino acid sequences to changes are described. Those skilled in the art are familiar with which amino acid changes can be tolerated at certain positions in the protein. For example, most buried amino acid residues require a non-polar side chain, whereas in general, surface side chain features are hardly conserved. Other such phenotypically silent substitutions are described in Bowie et al., Supra, and the references cited therein.

  Well-known and commonly available DNA sequencing methods can be used to implement any polynucleotide embodiment of the present invention. The methods include Klenow fragment of DNA polymerase I, SEQUENASE® (US Biochemical Corp, Cleveland, Ohio), Taq polymerase (Invitrogen), thermostable T7 polymerase (Amersham, Chicago, Ill.), Or recombinant polymerase And enzymes such as in combination with proofreading exonucleases such as ELONGASE Amplification System sold by Gibco BRL (Gaithersburg, Md.). The process can be automated using machines such as Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, Mass.) And ABI 377 DNA sequencer (Applied Biosystems).

  Polynucleotide sequences encoding the binding agents or antibodies of the invention are extended using partial nucleotide sequences and using various methods known in the art for detecting upstream sequences such as promoters and regulatory elements. obtain. For example, one method may be to use “restriction site” PCR that uses universal primers to search for unknown sequences adjacent to a known locus (Sarkar, G., PCR Methods Applic. 2: 318- 322 (1993)). In particular, genomic DNA is first amplified in the presence of primers for the linker sequence and primers specific for the known region. Exemplary primers are described in Example 4 herein. The amplified sequence is then subjected to a second round of PCR using the same linker primer and another specific primer inherent in the first primer. The product of each round of PCR is transcribed using an appropriate RNA polymerase and sequenced using reverse transcriptase.

  Inverse PCR can also be used (Triglia et al., Nucleic Acids Res. 16: 8186 (1988)) to amplify or extend sequences using a wide variety of primers based on known regions. Primers have a GC content of 50% or more, such as being 22-30 nucleotides in length using OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Plymouth, Minn.), Or another suitable program And can be designed to anneal to the target sequence at a temperature of about 68-72 ° C. The method uses several restriction enzymes to produce appropriate fragments in known regions of the gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.

  Another method that can be used is capture PCR, which performs PCR amplification of DNA fragments flanking known sequences in human and yeast artificial chromosome DNA (Lagerstrom et al., PCR Methods Applic. 1: 111-119 (1991)). . In this method, multiple restriction enzyme digests and ligations can also be used to place the processed double stranded sequence in an unknown part of the DNA molecule prior to performing PCR. Another method that can be used to search for unknown sequences is that described in Parker et al., Nucleic Acids Res. 19: 3055-3060 (1991). In addition, PCR, nested primers, and a PROMOTERFINDER ™ library (Clontech, Palo Alto, Calif.) For walking in genomic DNA can be used. This process is useful for discovering intron / exon junctions without having to screen the library.

  When screening full-length cDNAs, a library selected by size may be used to contain larger cDNAs, or randomly initiated containing more sequences including the 5 'region of the gene A library may be used. Randomly initiated libraries are useful in situations where an oligo d (T) library does not give a full-length cDNA. Genomic libraries can be useful for extension of sequences into 5 ′ and 3 ′ non-transcribed regulatory regions.

  Commercially available capillary electrophoresis systems can be used to analyze size or to verify the nucleotide sequence of sequencing or PCR products. In particular, capillary sequencing uses a flowable polymer for electrophoretic separation, four different fluorescent dyes activated by a laser (one for each nucleotide), and detection of the emission wavelength by a charge coupled device camera. obtain. Output / light intensity can be converted to electrical signals using appropriate software (e.g. GENOTYPER (TM) and SEQUENCE NAVIGATOR (TM), Applied Biosystems), from sample loading to computer analysis and electronic data display The entire process up to can be computer controlled. Capillary electrophoresis is useful for sequencing small pieces of DNA that can be present in a limited amount in a particular sample.

  The present invention provides a recombinant vector (e.g., an expression vector) comprising an isolated polynucleotide of the present invention, a host cell introduced with the recombinant vector (i.e., comprising the polynucleotide and / or containing the polynucleotide). Also provided is the production of recombinant binding agent polypeptides (eg, antibodies) or fragments thereof by recombinant techniques.

  As used herein, a “vector” is any construct that can deliver one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An “expression vector” can deliver and express one or more polynucleotides of interest as a polypeptide encoded in a host cell into which the expression vector has been introduced. Accordingly, the polynucleotide of interest in the expression vector is in the vector, or at or near or on the integration site of the polynucleotide of interest, such that the polynucleotide of interest is translated in the host cell into which the expression vector has been introduced. In any of the host cell's genome, it is operably linked to regulatory elements such as promoters, enhancers, poly A tails, to position expression in the vector. “Introduced” refers to electroporation, fusion with vector-containing liposomes, chemical transfection (eg, DEAE-dextran), transformation, transfection, and infection and / or transduction (eg, by recombinant viruses). ) Means that the vector is inserted into the host cell by any means, but not limited thereto. Thus, examples of vectors include viral vectors (which can be used to make recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors linked to cationic condensing agents. However, it is not limited to these.

  In some embodiments, a polynucleotide of the invention (e.g., encoding an EGFR mutant-specific binding agent) may be involved in the use of a non-pathogenic (deficient), replication competent virus, or replication. It can be introduced using a viral expression system that can use defective viruses (eg, urticaria or other poxviruses, retroviruses, or adenoviruses). In the latter case, viral propagation generally occurs only in complementary viral packaging cells. Suitable systems are described, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86: 317-321; Flexner et al., 1989, Ann. NY Acad Sci. 569: 86-103; Flexner et al., 1990. Vaccine 8: 17-21; U.S. Pat. WO 91/02805; Berkner-Biotechniques 6: 616-627, 1988; Rosenfeld et al., 1991, Science 252: 431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA 91: 215-219 Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA 90: 11498-11502; Guzman et al., 1993, Circulation 88: 2838-2848; and Guzman et al., 1993, Cir. Res. 73: 1202-1207 Is disclosed. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. The DNA may be “naked” as described, for example, in Ulmer et al., 1993, Science 259: 1745-1749 and discussed in Cohen, 1993, Science 259: 1691-1692. Naked DNA incorporation can be increased by coating the DNA on biodegradable beads that are efficiently transported into the cell.

  The polynucleotide can be linked to a vector containing a selectable marker for growth in the host. Usually, the plasmid vector is introduced into a precipitate, such as a calcium phosphate precipitate, or into a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells. The present invention can be practiced with vectors comprising cis-acting regulatory regions for the polynucleotide of interest. Appropriate trans-acting factors may be supplied by the host, supplied by a complementary vector, or supplied by the vector itself upon introduction into the host. In certain embodiments in this regard, the vector provides for specific expression that can be inducible and / or cell type specific (e.g., inducible by environmental factors that are easy to manipulate, such as temperature and nutrient additives). provide.

  A DNA insert comprising a polynucleotide encoding an antibody of the invention or encoding a binding agent may include, for example, the phage λPL promoter, the E. coli lac, trp and tac promoters, SV40 early and late, to name a few. It should be operably linked to a suitable promoter, such as a promoter, as well as the promoter of a retroviral LTR. Other suitable promoters are known to those skilled in the art. The expression construct further contains a transcription start site and a stop site, and a ribosome binding site for translation in the transcription region. The coding portion of the mature transcript expressed by the construct may include a translation and stop codon (UAA, UGA or UAG) starting from a start codon appropriately placed at the end of the polypeptide to be translated.

  As described, the expression vector may include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culture in E. coli and other bacteria. . Representative examples of suitable hosts include bacterial cells such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells such as yeast cells; Drosophila S2 and Insect cells such as Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes malignant melanoma cells; and plant cells, but are not limited to these. Appropriate media and conditions for the above host cells are known in the art.

  Vectors for use in bacteria include pQE70, pQE60 and pQE-9 available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A available from Stratagene; and available from Pharmacia Examples include, but are not limited to, ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5. Eukaryotic vectors include, but are not limited to, pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to those skilled in the art.

  Bacterial promoters suitable for use in the present invention include, but are not limited to, E. coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoter, λPR and PL promoters, and trp promoters. Suitable eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, retroviral LTR promoter such as Rous sarcoma virus (RSV) promoter, and metallothionein such as mouse metallothionein-I promoter A promoter is mentioned.

  In the yeast, Saccharomyces cerevisiae, several vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For a review, see Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, and Grant et al., Methods Enzymol. 153: 516-544 (1997).

  Introduction of the construct into the host cell can be performed by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other methods . Such methods are described in many standard laboratory manuals such as Davis et al., Basic Methods In Molecular Biology (1986).

  Transcription of the DNA encoding the binding agent or antibody of the invention by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10-300 bp, that act to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer (100-270 base pairs late in the origin of replication), the cytomegalovirus early promoter enhancer, the late polyoma enhancer in the origin of replication, and the adenovirus enhancer.

  Appropriate secretion signals can be incorporated into the expressed polypeptide to secrete the translated protein into the lumen of the endoplasmic reticulum or into the extracellular environment. The signal may be endogenous to the polypeptide or a signal of a different species.

  Polypeptides (eg, binding agents or antibodies) can be expressed in modified forms such as fusion proteins (eg, GST fusions) and can include not only secretion signals, but also additional heterologous functional regions. For example, additional amino acids, particularly charged amino acid regions, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell during purification or after purification and storage. A peptide moiety can also be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. In particular, adding a peptide moiety to a polypeptide for secretion or excretion, to improve stability, and to facilitate purification is a routine technique familiar in the art.

  By way of example, but not limitation, a binding agent or antibody of the invention may comprise a heterologous region derived from an immunoglobulin useful for solubilizing proteins. For example, EP-A-O 464 533 (Canadian counterpart 2045869) discloses a fusion protein comprising various portions of the constant region of an immunoglobulin molecule together with another human protein or a part thereof. In many cases, the Fc portion of the fusion protein is completely advantageous for use in therapy and diagnosis, thus, for example, leading to improved pharmacokinetic properties (EP-A 0232 262). On the other hand, for some uses it is desirable to be able to delete the Fc portion after expressing, detecting and purifying the fusion protein in the advantageous manner described. This is true if the Fc moiety has been found to interfere with the use in therapy and diagnosis, for example, if the fusion protein is to be used as an antigen for immunization. In drug discovery, human proteins such as, for example, hIL-5 are fused to the Fc portion for high information screening assays that identify hIL-5 antagonists. See Bennett et al., Journal of Molecular Recognition 8: 52-58 (1995) and Johanson et al., The Journal of Biological Chemistry 270 (16): 9459-9471 (1995).

  Binding agents and antibodies are well known, such as ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Can be recovered and purified from recombinant cell cultures. In some embodiments, high performance liquid chromatography (“HPLC”) is used for purification. The polypeptides of the present invention are naturally purified products, products of chemical synthesis procedures, and recombinantly produced in prokaryotic or eukaryotic hosts such as bacteria, yeast, higher plants, insects and mammalian cells. Products. The polypeptides of the present invention may or may not be glycosylated depending on the host used in the recombinant production procedure. Furthermore, the polypeptides of the present invention may also include a modified initiating methionine residue in some instances as a result of a host-mediated process.

  Accordingly, in another embodiment of the invention, a recombinant binding agent is obtained by culturing a recombinant host cell (as described above) and recovering the polypeptide under conditions suitable for expression of the fusion polypeptide. Alternatively, a method for producing an antibody is provided. Culture conditions suitable for growth of host cells and the expression of recombinant polypeptides from such cells are well known to those skilled in the art. See, for example, Current Protocols in Molecular Biology, Ausubel FM et al., Volume 2, Chapter 16, Wiley Interscience.

  The present invention also provides binding agents (particularly antibodies) that specifically bind to an epitope on a target molecule. Similarly, the present invention provides epitopes useful for identifying binding agents that specifically bind to target molecules that have the epitope. For example, an epitope comprising a threonine-serine-proline sequence (in the N-terminal to C-terminal direction) as described herein, in particular, is an epidermal growth factor receptor (EGFR) that is deficient at position E746-A750. Useful for identifying antibodies that specifically bind to a molecule.

Epitope mapping can be performed using standard methods. For example, phage display is an in vitro selection technique in which a peptide is genetically fused with a bacteriophage coat protein to display the fusion protein outside the virion. Biopanning of these virions by incubating the phage pool displayed a variant with the specific antibody of interest immobilized on the plate. Unbound phage are then washed, after which specific bound phage are eluted. The eluted phage are then amplified in E. coli and the process is repeated to enrich the phage pool in favor of the tightest binding sequences. The advantage of this technique is that it allows an unbiased screening of over 10 ⁹ sequences. Phage display is particularly useful when the immunogen is an unknown or high protein fragment.

  One of the limitations of phage display is cross-staining between phage particles. Cross-staining between phage particles can enrich sequences that do not specifically bind to antibodies. In addition, there are non-naturally occurring sequences in phage display peptide libraries. These sequences may not be at all similar to the immunizing peptide and may be tightly bound to the antibody of interest. Searching for sequences that do not resemble immunizing peptides can be very confusing, and it is difficult to decipher whether these peptides are contaminating or are non-natural peptides with high binding affinity for the antibody of interest It is.

The binders of the present invention can be used in a variety of ways. For example, the binding agents of the invention can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987). The binding agent may be provided with a detectable label (eg, a fluorophore such as FITC or phycoerythrin, or an enzyme substrate such as horseradish peroxidase substrate) to facilitate detection in the use of in vitro assays. As discussed below, the binding agents of the invention can be used in in vivo diagnostic assays, such as in vivo imaging. In some embodiments, the antibody is a radionuclide (such as ³ H, ¹¹¹ In, ¹⁴ C, ³² P, or ¹²³ I) so that immunoscintigraphy can be used to localize the cells or tissues of interest. Be labeled. Methods for binding labels to binding agents (such as antibodies) are known in the art. In other embodiments of the invention, the binding agent of the invention need not be labeled, and the presence of the binding agent of the invention can be detected using a labeled antibody that binds to the binding agent of the invention. The antibody can also be used as a staining reagent in lesions according to techniques well known in the art.

  The present invention also provides immortalized cell lines that produce the antibodies of the present invention. For example, hybridoma clones that produce monoclonal antibodies directed against the target sites disclosed herein, configured as described above, are also provided. Similarly, the present invention includes recombinant cells that produce the antibodies of the present invention, which cells can be constructed by well-known techniques, for example, the antigen binding site of a monoclonal antibody can be found in PCR as well as in E. coli. It can be cloned by phage-displayed recombinant antibodies or single chain antibodies made as soluble antibodies (see, eg, Antibody Engineering Protocols, 1995, Humana Press, Sudhir Paul).

  In another aspect of the present invention, a method for producing a specific antibody is provided.

  As described in detail below, the polyclonal antibody of the present invention immunizes an appropriate animal (eg, rabbit, goat, etc.) according to standard techniques, collects immune serum from the animal, and collects polyclonal antibody from the immune serum. Can be produced by screening and isolating polyclonal antibodies specific for the site of interest according to known procedures. Methods for immunizing non-human animals such as mice, rats, sheep, goats, pigs, cows and horses are well known in the art. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990.

  The immunogen can be a full-length protein or peptide containing the site of interest. In some embodiments, the immunogen is a peptide 7-20 amino acids in length, eg, about 8-17 amino acids in length. Peptide antigens suitable for antibody production of the invention can be designed, constructed and used according to well-known techniques. For example, Antibodies: A Laboratory Manual, Chapter 5, p. 75-76, edited by Harlow & Lane, Cold Spring Harbor Laboratory (1988); Czernik, Methods In Enzymology, 201: 264-283 (1991); Merrifield, J. Am Chem. Soc. 85: 21-49 (1962)).

  In some embodiments, the immunogen is administered in combination with an adjuvant. Suitable adjuvants will be well known to those skilled in the art. Typical adjuvants include Freund's complete or incomplete adjuvant, RIBI (muramyl dipeptide) or ISCOM (immunostimulatory complex).

  After the immunization, when the above method is used for the production of a polyclonal antibody, the polyclonal antibody secreted into the bloodstream can be recovered using a known technique. These purified antibody forms can, of course, be readily prepared by standard purification techniques (eg, affinity chromatography with protein A, anti-immunoglobulin, or the antigen itself). In either case, in order to monitor the establishment of immunization, the amount of antibody against serum antigen is monitored using standard techniques such as ELISA and RIA.

  The monoclonal antibodies of the invention can be produced by any of several methods well known in the art. In some embodiments, antibody-producing B cells are isolated from animals immunized with peptide antigens as described above. The B cells can be derived from spleen, lymph nodes or peripheral blood. Individual B cells are isolated and screened as described below to identify cells that produce the antibody of interest. Subsequently, the identified cells are cultured to produce the monoclonal antibody of the present invention.

  Alternatively, monoclonal antibodies of the invention can be produced using standard hybridoma technology in hybridoma cell lines according to the well-known techniques of Kohler and Milstein. See Nature 265: 495-97 (1975); Kohler and Milstein, Eur. J. Immunol. 6: 511 (1976). See also Current Protocols in Molecular Biology, edited by Ausubel et al. (1989). The monoclonal antibodies produced in this way are highly specific and improve the selectivity and specificity of the diagnostic assay methods provided by the present invention. For example, a solution containing the appropriate antigen can be injected into a mouse or other species, and after a sufficient amount of time (equivalent to conventional techniques), the animal can be sacrificed and splenocytes can be obtained. The splenocytes are then immortalized by any of several standard methods. Cell immortalization methods include transfection with oncogenes, cell infection with oncogenic viruses and cell culture under conditions selected for immortalized cells, placing cells under the influence of carcinogenic or mutated compounds, Examples include, but are not limited to, cell fusion with immortalized cells (eg, myeloma cells) and inactivation of tumor suppressor genes. See, for example, Harlow and Lane mentioned above. When used for fusion with myeloma cells, the myeloma cells preferably do not secrete immunoglobulin polypeptides (non-secreting cell lines). Typically, the cell-producing antibody to be fused and the immortalized cell (such as but not limited to myeloma cells) are of the same type. For example, a rabbit fusion hybridoma can be produced as described in C. Knight, US Pat. No. 5,675,063, issued Oct. 7, 1997. Cell-producing immortalized antibodies (such as hybridoma cells) are then grown in an appropriate selective medium such as hypoxanthine-aminopterin-thymidine (HAT), and monoclonal antibodies with the desired specificity are then isolated as described below. Screening Qing. Secreted antibodies can be recovered from the tissue culture supernatant by conventional methods such as precipitation, ion exchange or affinity chromatography.

  The present invention also includes antibody-producing cells and cell lines (such as hybridomas) as described above.

  Polyclonal or monoclonal antibodies can also be obtained by in vitro immunization. For example, phage display technology can be used to provide a library containing antibody repertoires with varying affinities for specific antigens. Techniques for identifying high affinity human antibodies from such libraries are described by Griffiths et al., (1994) EMBO J., 13: 3245-3260; Nissim et al., Ibid, pp. 692-698 and Griffiths, incorporated by reference. Et al., Ibid, 12: 725-734. Antibodies can be produced recombinantly using methods well known in the art, for example, according to the methods disclosed in US Pat. No. 4,349,893 (Reading) or US Pat. No. 4,816,567 (Cabilly et al.). Antibodies can also be chemically constructed with specific antibodies made by the methods disclosed in US Pat. No. 4,676,980 (Segel et al.).

  Once the desired antibody is identified, an antibody comprising heavy chain, light chain, or both (or single chain in the case of single chain antibodies) or a portion thereof using methods well known in the art Can be cloned and isolated from antibody producing cells (eg, polynucleotides encoding variable regions, etc.). For example, the antigen binding site of a monoclonal antibody can be cloned by PCR and single-chain antibodies made as phage-displayed recombinant antibodies or soluble antibodies in E. coli (see, for example, Antibody Engineering Protocols, 1995, (See Humana Press, Sudhir Paul).

  Accordingly, in a further aspect of the invention there is provided such a polynucleotide encoding the heavy chain, light chain, variable region, framework region or CDR of the antibody of the invention. In some embodiments, the nucleic acid is operably linked to an expression control sequence. Accordingly, the present invention also provides vectors and expression control sequences useful in recombinant expression of the antibodies of the invention or antigen binding portions thereof. Those skilled in the art will be able to select vectors and expression systems suitable for the host cell in which the antibody or antigen-binding portion is expressed.

  The monoclonal antibodies of the invention can be produced recombinantly by expressing the encoding nucleic acid in a suitable host cell under suitable conditions. Accordingly, the present invention further provides a host cell comprising the above nucleic acid and vector.

  Monoclonal Fab fragments may be produced in Escherichia coli by recombinant techniques known to those skilled in the art. See, for example, W. Huse, Science 246: 1275-81 (1989); Mullinax et al., Proc. Nat'l Acad. Sci. 87: 8095 (1990).

  If a monoclonal antibody of one desired isotype is preferred for a particular application, it can be prepared directly by selecting a particular isotype from the initial fusion or sibling selection techniques for isolating class switch variants. It can be prepared side-by-side from parental hybridomas that secrete monoclonal antibodies of different isotypes by use (Steplewski et al., Proc. Nat'l. Acad. Sci., 82: 8653 (1985); Spira et al., J. Immunol. Methods, 74: 307 (1984)). Alternatively, isotypes of monoclonal antibodies having desired characteristics can be changed using antibody production techniques well known in the art.

  The antibodies of the invention, whether polyclonal or monoclonal, can be screened for epitope specificity according to standard techniques. See, for example, Czernik et al., Methods in Enzymology, 201: 264-283 (1991). Peptide competition assays can be performed to confirm that they do not react with other epitopes. Antibodies can also be tested by Western blotting in cell preparations containing the parent signaling protein (e.g., cell lines overexpressing the parent protein) to confirm the reactivity of the antibody with the desired epitope / target. Good.

In an exemplary embodiment, a phage display library containing more than 10 ¹⁰ phage clones is used for mass production of monoclonal antibodies, and high information is used to screen the efficacy of these antibodies in validation and quality control. A quantity of immunohistochemistry is used. Western blots, protein microarrays and flow cytometry can also be used in high information screening of site-specific polyclonal or monoclonal antibodies of the invention. See, for example, Blow N., Nature, 447: 741-743 (2007).

  The antibodies of the present invention may exhibit some limited cross-reactivity with related epitopes in non-target proteins. This is not unexpected since most antibodies show some degree of cross-reactivity, and anti-peptide antibodies often cross-react with epitopes that have a high homology with the immunizing peptide. For example, see Czernik mentioned above. Cross-reactions with non-target proteins are easily characterized by Western blotting using markers with known molecular weights.

  In certain cases, polyclonal antisera may exhibit some undesirable general cross-reactions that can be removed by further purification of the antisera (eg, on a phosphotyrosine column).

  The nature of the antibody can be further determined by immunohistochemistry (IHC) staining using normal and diseased tissue. IHC can be performed according to well-known techniques. See, for example, Antibodies: A Laboratory Manual, Chapter 10, Harlow & Lane, Cold Spring Harbor Laboratory (1988). Briefly, paraffin-embedded tissue for immunohistochemical staining (eg, tumor tissue) is deparaffinized tissue sections with xylene followed by ethanol; hydrated in water then PBS; sodium citrate Expose antigen by heating slides in buffer; incubate in hydrogen peroxide; block in blocking solution; incubate slides in primary and secondary antibodies; finally, manufacturer's instructions According to the detection using the ABC avidin / biotin method.

  The nature of the antibody can be further determined by flow cytometry performed according to standard methods. See Chow et al., Cytometry (Communications in Clinical Cytometry) 46: 72-78 (2001). Briefly, by way of example, the following protocol for cytometric analysis may be used. That is, the sample can be centrifuged on a Ficoll gradient to remove lysed red blood cells and cell debris. From the plate, the attached cells can be scraped and washed with PBS. Cells can then be fixed with 2% paraformaldehyde at 37 ° C. for 10 minutes and then permeabilized in 90% methanol on ice for 30 minutes. The cells can then be stained with a primary antibody of the invention, washed and labeled with a fluorescently labeled secondary antibody). At this time, a marker antibody (eg, CD45, CD34) bound with an additional fluorescent dye may be added to assist in the identification of the subsequent specific blood cell type. The cells are then analyzed on a flow cytometer (eg Beckman Coulter FC500) according to the instrument specific protocol used.

  The binding agents of the present invention can also be used with fluorescent dyes (e.g. Alexa488, PE) for use in multiparametric analysis with other signaling (phospho-CrkL, phospho-Erk1 / 2) and / or cell marker (CD34) antibodies. It can be advantageously combined. Methods for making bispecific antibodies are within the purview of those skilled in the art. Conventionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305 : 537-539 (1983)). Antibody variable regions (antibody antigen binding sites) having the desired binding specificities can be fused to immunoglobulin constant region sequences. In certain embodiments, the fusion with an immunoglobulin heavy chain constant region comprises at least a portion of the hinge, CH2, and CH3 regions. The immunoglobulin heavy chain fusion and, if desired, the DNA encoding the immunoglobulin light chain are inserted into a separate expression vector and cotransfected into a suitable host organism. For details illustrating methods for making bispecific antibodies known at present, see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986); WO 96/27011; Brennan et al., Science 229: 81. (1985); Shalaby et al., J. Exp. Med. 175: 217-225 (1992); Kostelny et al., J. Immunol. 148 (5): 1547-1553 (1992); Hollinger et al., Proc. Natl. Acad. See Sci. USA 90: 6444-6448 (1993); Gruber et al., J. Immunol. 152: 5368 (1994); and Tutt et al., J. Immunol. 147: 60 (1991). Bispecific antibodies also include cross-linked or heteroconjugate antibodies. Heteroconjugate antibodies can be made using any convenient crosslinking method. Suitable crosslinkers are well known in the art and are disclosed in US Pat. No. 4,676,980 along with several crosslinking techniques.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148 (5): 1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins can bind to Fab ′ portions of two different antibodies by gene fusion. The antibody homodimer can be reduced at the hinge region to form a monomer and then reoxidized to form an antibody heterodimer. This method can also be used to produce antibody homodimers. A strategy for producing bispecific antibody fragments using single chain Fv (scFv) dimers has also been reported. See Gruber et al., J. Immunol., 152: 5368 (1994). Alternatively, the antibody may be a “linear antibody” as described in Zapata et al. Protein Eng. 8 (10): 1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (V _H -C _H 1 -V _H -C _H 1) that form a pair of antigen binding regions. Linear antibodies may be bispecific or monospecific.

  To produce a chimeric antibody, portions derived from two different species (e.g., human constant region and mouse variable or binding region) can be chemically linked by conventional techniques, or one using genetic engineering techniques. Can be prepared as a continuous protein. DNA molecules encoding both the light and heavy chain proteins of the chimeric antibody can be expressed as a continuous protein. Methods for making chimeric antibodies are disclosed in US Pat. No. 5,677,427, US Pat. No. 6,120,767, and US Pat. No. 6,329,508, each of which is incorporated by reference in their entirety.

  Fully human antibodies can be produced by a variety of techniques. One example is the trioma method. For the basic approach and SPAZ-4, a representative cell fusion partner for use in this approach, see Oestberg et al. Hybridoma 2: 361-367 (1983); Oestberg US Pat. No. 4,634,664; and Engleman et al. U.S. Pat. No. 4,634,666, each of which is incorporated by reference in its entirety.

  Human antibodies can also be produced from non-human transgenic animals having a transgene encoding at least a segment of the human immunoglobulin locus. The production and characteristics of animals having these characteristics are described, for example, in Lonberg et al., WO 93/12227; US Pat. No. 5,545,806; and Kucherlapati et al., Which are incorporated herein by reference in their entirety. Reference is made to publication 91/10741; U.S. Pat. No. 6,150,584.

  Various recombinant antibody library techniques can also be used to produce fully human antibodies. For example, one approach includes screening a human B cell-derived DNA library according to the general protocol outlined in Huse et al., Science 246: 1275-1281 (1989). The protocol described in Huse has been shown to be more efficient when used in conjunction with phage display technology. See, for example, Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047, US Pat. No. 5,969,108, each of which is incorporated by reference in its entirety.

  Methods for displaying antibody libraries and isolating human antibody binding are as follows: Coia G et al., J. Immunol. Methods 1: 254 (1-2): 191-7 (incorporated in their entirety by reference). 2001); Hanes J. et al., Nat. Biotechnol. 18 (12): 1287-92 (2000); Proc. Natl. Acad. Sci. USA 95 (24): 14130-5 (1998); Proc. Natl. Acad Eukaryotic ribosomes can also be used by screening for target antigens, as described in Sci. USA 94 (10): 4937-42 (1997).

  Yeast systems are also suitable for screening of mammalian cell surfaces or secreted proteins (such as antibodies). Antibody libraries can be displayed on the surface of yeast cells for the purpose of obtaining human antibodies against the target antigen. This approach is described in Yeung et al., Biotechnol. Prog. 18 (2): 212-20 (2002); Boeder, ET et al., Nat. Biotechnol. 15 (each of which is incorporated herein by reference in its entirety. 6): 553-7 (1997). Alternatively, human antibody libraries can be expressed in cells and screened through a yeast two-hybrid system (WO 0200729A2, incorporated in its entirety by reference).

  Any of the recombinant specific antibodies described herein, and any, including both prokaryotic and eukaryotic expression systems such as bacteria, yeast, insect cells, plant cells, mammalian cells (eg, NS0 cells) Recombinant DNA technology can be used to produce chimeric or humanized antibodies or any other genetically modified antibody and fragments or conjugates thereof in the expression systems of

  Once produced, the complete antibodies of the present application, dimers of the complete antibodies, individual light and heavy chains or other immunoglobulin forms, such as ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, etc. Purification can be done according to standard procedures in the art (see generally Scopes, R., Protein Purification (Springer-Verlag, NY, 1982)). The polypeptide once purified partially or homogeneously as desired can then be used in therapy (including in vitro) or in developing and performing assay procedures, immunofluorescent staining, etc. Immunological Methods, Vols. I and II (see Lefkovits and Pernis, Academic Press, NY, 1979 and 1981).

  In another aspect of the present invention, a method for identifying cancers in which EGFR targeted therapy works well is provided. The method comprises: (a) a binding agent that specifically binds to either an epidermal growth factor receptor (EGFR) molecule lacking position E746-A750 or an EGFR molecule having an L858R point mutation; and a biological sample from cancer And (b) the amount of binding obtained by contacting a biological sample derived from a healthy individual with a binding agent, and (b) And the step of showing the binding amount that the EGFR targeted therapy responds well to the cancer by the difference between the binding amount and the cancer-derived binding amount.

  "EGFR targeted therapy" refers to EGFR molecules (or mutants of EGFR molecules such as L858R mutant or E746-A750del mutant) and presents with cancer characterized by EGFR abnormal expression or EGFR abnormal A compound that inhibits physical (e.g., surgical) or pharmaceutical (e.g., EGFR expression and / or activity) for a patient or patient (e.g., a human patient) suspected of being susceptible to a cancer characterized by expression ) Means any treatment whether or not.

  As used herein, "EGFR abnormal expression" in one individual or tissue refers to overexpression or low expression of wild-type EGFR and / or a mutant form of a molecule in the tissue compared to the same tissue of an unaffected individual Means expression. For example, tissue expression of an EGFR mutant (eg, EGFR L858R mutant or E746-A750del mutant) is an abnormal expression of EGFR in the tissue. Similarly, if an individual expresses EGFR molecules in a tissue while EGFR is not expressed or expressed in different amounts in the same tissue type of a healthy individual, the individual is said to be abnormally expressing EGFR .

  In some embodiments, the cancer is from a human patient. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the cancer is adenocarcinoma or squamous cell carcinoma. In some embodiments, the cancer is a tissue type selected from the group consisting of lung cancer, colon cancer, breast cancer, cervical cancer, pancreatic cancer, prostate cancer, gastric cancer, and esophageal cancer.

  In various embodiments, the tissue type of a cancer-derived biological sample and a healthy individual-derived biological sample are the same. Of course, a biological sample derived from cancer is, of course, neoplastic (malignant or benign), but a biological sample derived from a healthy individual may have the same tissue type as the biological sample derived from cancer. For example, when the cancer is NSCLC, the biological sample derived from a healthy individual can be a lung tissue sample. Similarly, when the cancer is a pancreatic adenocarcinoma, the biological sample from a healthy individual can be a pancreatic tissue sample.

`` Perform well '' means a reduction in cancer (which may be benign or malignant) after molecular targeted therapy (e.g. EGFR mutant targeted therapy) (e.g. in the case of solid tumors), a decrease in the number of neoplastic cells ( For example, it means non-solid tumors such as leukemia), non-increased size (eg, for solid tumors), or non-increased number of new cells (eg, for non-solid tumors). The number of cancer cells can be measured in a blood sample using, for example, a hemocytometer. For solid tumors, the size can be determined using a caliper, and if the tumor has been excised, it can be determined by weighing the tumor on a scale.

  As used herein, the term “biological sample” or “tissue sample” is used in its broadest sense, and any living organism suspected of containing a molecule of interest (eg, an EGFR molecule or a mutant thereof). Means sample, cell, chromosome isolated from the cell (e.g. metaphase chromosome spread), genomic DNA (in solution or combined with a solid support such as Southern analysis), in solution or in Northern It can include extracts such as RNA (coupled to a solid support such as analysis), cDNA (in solution or coupled to a solid support), cells, blood, urine, spinal fluid, or tissue.

  A biological sample useful in the practice of the methods of the invention may be obtained from any mammal that exhibits, or is likely to develop, a cancer characterized by the presence of the molecule of interest. As used herein, the phrase "characterized by" with respect to cancer and specified molecules (e.g., EGFR aberrant expression (e.g., EGFR overexpression or EGFR mutant expression)) does not cause the specified molecule to aberrantly express It means a cancer in which a designated molecule is abnormally expressed as compared to a neoplastic or non-neoplastic biological sample of the same tissue type. The presence of aberrantly expressed EGFR may completely (or stimulate or become a pathogenic agent) the growth and survival of such cancers.

  Any biological sample containing cells (or cell extracts) obtained from mammalian cancer is suitable for use in the methods of the invention. In one embodiment, the biological sample comprises cells obtained from a tumor biopsy. A biopsy may be obtained according to standard clinical techniques, from a primary tumor present in a mammalian organ or by a secondary tumor that has metastasized into other tissues. In another embodiment, the biological sample comprises cells obtained from fine needle aspirate collected from a tumor, and techniques for obtaining such aspirate are well known in the art (Cristallini et al., Acta Cytol. 36 ( 3): see 416-22 (1992)).

  The cell extracts of the above biological samples can be prepared unpurified or partially (or fully) purified and used in the methods of the invention according to standard techniques. Alternatively, biological samples containing complete cells can be used in assay formats such as immunohistochemistry (IHC), flow cytometry (FC), and immunofluorescence (IF). Such full cell assays minimize the manipulation of tumor cell samples and thus reduce the risk of altering the cell's in vivo signaling / activation state and / or inducing artificial signals This is advantageous. Complete cell assays are also advantageous because they characterize expression and signal transduction only in tumor cells, not a mixture of tumor cells and normal cells.

  As used herein, an “individual” (also referred to herein as a “subject”) or a “patient” or “patient” is a mammal (eg, a human, domestic animals (such as cows, pigs, and chickens), breeding Vertebrates such as animals (such as cats, parrots, turtles, lizards, dogs, and horses), primates (such as chimpanzees and gorillas), and zoo animals (such as but not limited to mammals) It is. The patient or patient may or may not be afflicted with a medical condition (eg, cancer) and / or may or may not currently present symptoms. In some embodiments, the subject exhibits cancer. In some embodiments, the subject has a tumor or has had a tumor removed. It is understood that even if the tumor has been removed from the subject, in some cases, tumor cells may remain in the subject despite being removed. For example, even if some of the tumors have been removed, the tumors may have spread and expanded to other parts of the body. Also, although the tumor can be removed from the subject, some or some tumor cells of the tumor may inadvertently or inevitably remain in the subject due to limitations of surgical procedures and the like. In some embodiments, the subject is at risk of developing a tumor (or cancer). In some embodiments, the subject is undergoing or is undergoing additional therapy (eg, chemotherapy, surgery, hormone therapy, radiation, or booster immunotherapy).

  Although the methods herein are primarily focused on the treatment of human subjects, the disclosed methods can also be used to treat other mammalian subjects such as dogs and cats for veterinary purposes.

  In some embodiments, a method of identifying cancer that is well responsive to EGFR targeted therapy may be performed prior to a preliminary blood test or a surgical investigation procedure. Such diagnostic assays can be used to identify patients or livestock that typically express EGFR in tissues where EGFR is not expressed in non-diseased individuals. Patients or patients with abnormal EGFR expression can be considered as patients or patients whose cancer or cancer development risk can be retained and EGFR targeted therapy may well respond.

  The method can be used, for example, in the case of a biological sample from a subject who has already been diagnosed with cancer and / or has not yet received cancer treatment, and the method can be used to diagnose a disease or monitor the progression of potential disease states. Used to help. For example, the method can be used when the target patient or patient has already been diagnosed with cancer, and optionally has already been treated for the disease, and the method can monitor disease progression involving EGFR abnormal expression. Used for.

  The methods of the present invention can also be used to assess the risk of developing cancer in a subject patient or livestock (eg, a patient or livestock that has a family history of cancer but has not yet shown symptoms).

  In another aspect of the present invention, there is provided a method for treating a patient or patient with cancer characterized by abnormal expression of EGFR or a patient or patient suspected of having the cancer, wherein the method is deficient at position E746-A750. A binding agent that specifically binds to either an epidermal growth factor receptor (EGFR) molecule or an EGFR molecule having an L858R point mutation, a polynucleotide encoding such a binding agent, a vector comprising such a polynucleotide, And / or administering a composition comprising the binding agent, the polynucleotide, or the vector to the patient or patient in an effective amount. In some embodiments, the cancer is characterized by EGFR aberrant expression.

  “Treatment” means the cessation, retarding or suppression of cancer progression in a patient or patient, or prevention of cancer development. In some embodiments, the cancer is a cancer characterized by the presence of molecules that specifically bind to the administered binding agent.

  In some embodiments, the subject aberrantly expresses an EGFR molecule (e.g., overexpressed or underexpressed wtEGFR, or an EGFR such as an EGFR L858R mutant or an EGFR E746-A750 deficient mutant described herein). A cancer that expresses a mutant molecule), or such a tumor has been removed and / or a biopsy of such a tumor has been taken. In some embodiments, tumor regression, reduced metastasis, and / or reduced tumor size or reduced tumor cell number encodes an effective amount of a binding agent (or composition comprising it) and / or binding agent. Induced by administration of a polynucleotide (or a composition comprising it).

  As used herein, an “effective amount” is beneficial such as pausing, delaying, pausing, retarding, or inhibiting cancer progression in a patient or patient, or preventing cancer development in a patient or patient An amount or dose sufficient to produce any or desired result. The effective amount depends on, for example, the binding agent, the polynucleotide encoding the binding agent, the age and weight of the subject to which the vector containing the polynucleotide and / or the composition thereof is administered, the severity of symptoms, and the route of administration. Because of differences, administration is determined on an individual basis. Usually, the daily dose for adults in the case of oral administration is about 0.1-1000 mg, administered as a single dose or divided doses. In the case of continuous intravenous administration, the composition can be administered in the range of 0.01 μg / kg / min to 1.0 μg / kg / min, desirably 0.025 μg / kg / min to 0.1 μg / kg / min.

  Thus, in a further aspect of the invention, the deletion of E746-A750, a binding agent that specifically binds to an epidermal growth factor receptor (EGFR) molecule having a point mutation in which leucine at position 858 is replaced by arginine. Also provided are binding agents that specifically bind to an epidermal growth factor receptor (EGFR) molecule, or a composition comprising both binding agents. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

  The present invention relates to a polynucleotide encoding a binding agent that specifically binds to an epidermal growth factor receptor (EGFR) molecule having a point mutation in which leucine at position 858 is replaced by arginine, and lacks E746-A750 position. Also provided are compositions comprising a polynucleotide encoding a binding agent that specifically binds to an epidermal growth factor receptor (EGFR) molecule, or both polynucleotides or vectors containing the same. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

  An effective amount of a binding agent (such as an antibody) of the invention, a polynucleotide encoding the binding agent, a vector containing such a polynucleotide, or a composition thereof can be administered in a single dose or multiple doses. As an example, an effective amount of a binding agent, such as an EGFR L858R mutant-specific antibody or an EGFR E746-A750del-specific antibody, may improve or stop progression of a patient or patient's condition (e.g., cancer characterized by EGFR abnormal expression), An amount sufficient to stabilize, reverse, slow and / or delay or improve proliferation, arrest, stabilization, retrograde, in vitro (e.g., biopsy cancer cells), An amount sufficient for slowing and / or delaying. As understood in the art, for example, an effective amount of an EGFR L858R variant-specific antibody or an EGFR E746-A750del-specific antibody includes, among other things, a history of the patient or patient, as well as the EGFR L858R variant-specific antibody used. Alternatively, it may vary depending on other factors such as the type (and / or dose) of the EGFR E746-A750del specific antibody.

  Effective amounts and schedules for administration of the binding agents, polynucleotides encoding the binding agents, and / or compositions of the invention can be determined empirically, and making such determinations is within the skill of the art. is there. The dosage to be administered is, for example, the binding agent of the invention, the polynucleotide encoding the binding agent, and / or the mammal receiving the composition, the route of administration, the binding agent of the invention used, the binding Those skilled in the art will appreciate that the polynucleotide encoding the agent and / or the particular type of composition and the other agent being administered to the mammal will vary. When administering an antibody and / or a composition comprising an antibody to a patient or patient, guidance in selecting an appropriate dose of the antibody can be found in literature on therapeutic use of antibodies, e.g., Handbook of Monoclonal Antibodies, edited by Ferrone et al., Noges Publications, Park Ridge, NJ, 1985, ch. 22 and pp. 303-357; found in Smith et al., Antibodies in Human Diagnosis and Therapy, edited by Haber et al., Raven Press, New York, 1977, pp. 365-389. .

  A typical daily dosage of an effective amount of a binder used alone may range from about 1 μg / kg body weight to 100 mg / kg body weight per day or more, depending on the factors described above. In general, the following doses: at least about 50 mg / kg body weight; at least about 10 mg / kg body weight; at least about 3 mg / kg body weight; at least about 1 mg / kg body weight; at least about 750 μg / kg body weight; at least about 500 μg administered at least about 250 μg / kg body weight; at least about 100 μg / kg body weight; at least about 50 μg / kg body weight; at least about 10 μg / kg body weight; at least about 1 μg / kg body weight; Can do. In some embodiments, the dose of a binding agent (e.g., antibody) provided herein is about 0.01 mg / kg to about 50 mg / kg, about 0.05 mg / kg to about 40 mg / kg, about 0.1 mg to about About 30 mg / kg, about 0.1 mg to about 20 mg / kg, about 0.5 mg to about 15 mg, or about 1 mg to 10 mg. In some embodiments, the dose is about 1 mg to 5 mg. In some alternative embodiments, the dose is about 5 mg to 10 mg.

  In some embodiments, the methods described herein further comprise a step of treating the subject with the additional therapy form, and / or the compositions described herein further comprise an additional agent for the additional therapy. . In some embodiments, the additional therapy form is an additional anticancer therapy (eg, the composition may include an anticancer agent). In some embodiments, the methods described herein further comprise treating the subject with chemotherapy, radiation, surgery, hormonal therapy, and / or booster immunotherapy. In some embodiments, the radiation is external beam radiation therapy or teletherapy. In some alternative embodiments, the radiation is delivered as internal therapy or brachytherapy. In some embodiments, the additional regimen comprises administration of one or several therapeutic agents (eg, kinase inhibitors, etc.). In some embodiments, the therapeutic agent is an anti-VEGF antibody Avastin®, an anti-HER2 antibody Herceptin® (Trastuzumab) (Genentech, Calif.), An anti-CD25 antibody Zenapax® (Daclizumab) (Roche Pharmaceuticals, Switzerland), and anti-CD20 antibody Rituxan® (IDEC Pharm./Genentech, Roche / Zettyaku).

  In some embodiments, the additional therapeutic agent is an angiogenesis inhibitor.

  In some embodiments, the additional therapeutic agent is a cytotoxic compound. In some embodiments, the binding agents of the invention can also be used to target cancer cells for effector-mediated cell death. For example, the binding agents (eg, antibodies) of the present invention can directly kill cancer cells through complement-mediated or antibody-dependent cell cytotoxicity. The binding agents (eg, antibodies) disclosed herein can be administered as a fusion molecule conjugated with a cytotoxic moiety to directly kill cancer cells. Fusion can be accomplished chemically or genetically (eg, via expression as a single fusion molecule). As will be appreciated by those skilled in the art, chemical fusion is used for small molecules, while for biological compounds, either chemical or genetic fusion can be used.

  Examples of cytotoxic compounds include, but are not limited to, therapeutic agents, radiation therapeutic agents, ribosome inactivating protein (RIP), chemotherapeutic agents, toxic peptides, toxic proteins, and mixtures thereof. Exemplary chemotherapeutic agents that can be combined with or included in the compositions of the invention include taxol, doxorubicin, docetaxel, prednisone, cisplatin, mitomycin, progesterone, tamoxifen, verapamil, podophyllotoxin , Procarbazine, mechloretamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, dactinomycin, daunorubicin, bleomycin, pricamycin, etoposide (VP16), transplatinum preparation, 5-fluorouracil, vincristine , Vinblastine, or methotrexate.

  In some embodiments, the additional therapeutic agent is an anti-inflammatory agent.

  The cytotoxic agent can be a cytotoxic agent that acts in the cell, such as a short range radiation emitter (eg, a short range, high energy alpha emitter). Enzymatically active toxins and fragments thereof (such as ribosome inactivating proteins) are represented by saporin, ruffin, momordin, ricin, trichosanthin, gelonin, abrin and the like. Procedures for preparing enzymatically active polypeptides of immunotoxins are described in WO 84/03508 and WO 85/03508, which are incorporated herein by reference. Certain cytotoxic moieties are derived from, for example, adriamycin, chlorambucil, daunomycin, methotrexate, neocalcinostatin, and platinum.

Alternatively, the binding agent can bind to a radioisotope of a high energy radiation emitter ( ¹³¹ I, γ emitter, etc.) that inactivates some cell diameters when locally irradiated to the tumor site. See, for example, SE Order, “Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy”, Monoclonal Antibodies for Cancer Detection and Therapy, Baldwin et al. (Ed.), Pp, incorporated herein by reference. See .303-316 (Academic Press 1985). Other suitable radioisotopes include α emitters such as ²¹² Bi, ²¹³ Bi, and ²¹¹ At, and β emitters such as ¹⁸⁶ Re and ⁹⁰ Y.

  The methods described herein (including therapeutic methods) and compositions described herein can be administered by a single direct injection at a single time point or multiple time points to a single or multiple sites. Administration can also be made to multiple sites almost simultaneously. The number of doses can be determined and adjusted throughout the course of therapy and is based on obtaining the desired result. In some instances, continuous sustained release formulations of binding agents (including antibodies), polynucleotides, and pharmaceutical compositions of the invention may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

  A binding agent (e.g., an antibody), a polynucleotide encoding the binding agent, and / or a vector comprising such a polynucleotide, or a composition comprising any of these, a carrier (e.g., a pharmaceutically acceptable carrier) Can be administered to patients or livestock. Accordingly, in a further aspect of the invention, a pharmaceutically acceptable carrier and (a) a binding agent of the invention, (b) a polynucleotide encoding the binding agent of the invention, and / or (c) encoding the binding agent. A composition (eg, pharmaceutical composition) comprising a vector comprising the polynucleotide to be produced.

  As used herein, “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” refers to a component that retains biological activity upon delivery when used in combination with an active ingredient, such that the subject's immune system Any material that does not react with and is non-toxic to the subject. Examples include, but are not limited to, any standard pharmaceutical carrier such as phosphate buffered saline solution, water, emulsions (such as oil / water emulsions), and various humectant types. Examples of diluents for aerosol or parenteral administration include, but are not limited to, phosphate buffered saline, normal (0.9%) saline, Ringer's solution and dextrose solution. The pH of the solution can be about 5 to about 8, or about 7 to about 7.5. Additional carriers include sustained release preparations such as solid hydrophobic polymer semipermeable matrices containing antibodies, which are in the form of molded articles, for example, fillers, liposomes or microparticles. . It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of antibody being administered. Compositions containing such carriers are well known in the art (e.g., Remington's Pharmaceutical Sciences, 18th edition, edited by A. Gennaro, Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000).

  While any suitable carrier known to those skilled in the art may be used in the pharmaceutical composition of the invention, the carrier type will depend on the method of administration. Rolland, 1998, Crit. Rev. Therap. Drug Carrier Systems 15: 143-198, and those described in references cited therein (i.e., binding agents or Numerous delivery techniques are well known in the art (including polynucleotides encoding binding agents).

  A composition comprising a binding agent of the invention and / or a polynucleotide encoding the binding agent ensures delivery of the composition to the bloodstream in an effective form, eg, systemic, topical, oral, transdermal. It may be formulated for any suitable method of administration, including nasal, intravenous, intracerebral, intraperitoneal, subcutaneous or intramuscular administration, or other methods such as infusion. The composition may also be administered by isolated perfusion techniques, such as isolated tissue perfusion, to exert a local therapeutic effect. Carriers for parenteral administration, such as subcutaneous injection, preferably include water, saline, alcohol, lipids, waxes or buffers. For oral administration, any of the above carriers or solid carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and magnesium carbonate may be used. In some embodiments, a formulation of a composition for oral administration is digested, for example, as a microcapsule encapsulating a binding agent (or a polynucleotide encoding the binding agent or a vector comprising such a polynucleotide) in a liposome. Withstands disassembly in the tube. Biodegradable microspheres (eg, polylactide-polyglycolide) can also be used as carriers in the pharmaceutical compositions of the present invention. Suitable biodegradable microspheres are disclosed, for example, in US Pat. Nos. 4,897,268 and 5,075,109.

  The composition of the present invention comprises a buffer (e.g., neutral buffered saline or phosphate buffered saline), carbohydrate (e.g., glucose, mannose, sucrose or dextran), mannitol, protein, polypeptide or Amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (eg, aluminum hydroxide) and / or preservatives may also be included. Alternatively, the compositions of the invention can be formulated as lyophilized.

  In some embodiments, the binding agent and / or the polynucleotide encoding the binding agent is, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly (methyl methacrylate) microcapsules, respectively. ) May be encapsulated in a prepared colloidal drug delivery system in microcapsules (eg, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or microemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 18th edition, edited by A. Gennaro, Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000. . To increase the serum half-life of the binding agent (eg, antibody), one may incorporate a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described in US Pat. No. 5,739,277, for example. As used herein, the term “salvage receptor binding epitope” is an epitope of the Fc region of an IgG molecule (eg, IgG1, IgG2, IgG3, and IgG4) that increases the in vivo serum half-life of the IgG molecule. Refers to something involved in

  The binding agents (and / or polynucleotides encoding the binding agents) disclosed herein may be formulated as liposomes. Liposomes containing a binding agent (and / or a polynucleotide encoding the binding agent) are described in Epstein et al., 1985, Proc. Natl. Acad. Sci. USA 82: 3688; Hwang et al., 1980, Proc. Natl Acad. Sci. USA 77: 4030; and US Pat. Nos. 4,485,045 and 4,544,545, and are prepared by methods known in the art. Liposomes with enhanced circulation time are disclosed in US Pat. No. 5,013,556. Particularly useful liposomes can be made by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a predetermined pore size filter to obtain liposomes having a desired diameter. Further, when the binding agent is an antibody, the antibody (including antigen binding region fragments such as Fab ′ fragments) can be obtained as described in Martin et al., 1982, J. Biol. Chem. 257: 286-288. Can bind to liposomes via a disulfide exchange reaction. Administration of expression vectors includes local or systemic administration, such as injection, oral administration, particle gun or catheter administration, and topical administration. Those skilled in the art are familiar with administering expression vectors to obtain exogenous protein expression in vivo. See, for example, US Pat. Nos. 6,436,908; 6,413,942; and 6,376,471.

  Targeted delivery of therapeutic compositions comprising a polynucleotide encoding a binding agent (eg, antibody) of the invention can also be used. Receptor-mediated DNA delivery techniques are described, for example, by Findeis et al., Trends Biotechnol. (1993) 11: 202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (JA Wolff) (1994); Wu et al., J Biol. Chem. (1988) 263: 621; Wu et al., J. Biol. Chem. (1994) 269: 542; Zenke et al., Proc. Natl. Acad. Sci. (USA) (1990) 87: 3655; Wu. Et al., J. Biol. Chem. (1991) 266: 338. The therapeutic composition containing the polynucleotide is administered in the range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. Concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA can also be used during gene therapy protocols. The therapeutic polynucleotides and polypeptides of the present invention can be delivered using gene delivery vehicles. Gene delivery vehicles may be of viral or non-viral origin (generally, Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5: 845; Connelly, Human Gene Therapy (1995 ) 1: 185; and Kaplitt, Nature Genetics (1994) 6: 148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence may be constitutive or regulatory.

  Viral-based vectors for delivery and expression of the desired polynucleotide in the desired cell are well known in the art. Exemplary virus-based vehicles include recombinant retroviruses (e.g., PCT WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. Pat.No. 5,219,740; U.S. Pat.No. 4,777,127; British Patent 2,200,651; and European Patent 0 345 242. See), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246), and Venezuelanoma. Encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-associated virus (AAV) vectors (e.g., PCT WO 94/12649, WO 93/03769 No .; WO 93/19191; WO 94/28938; see WO 95/11984 and WO 95/00655). However, it is not limited to these. Administration of DNA linked to killed adenovirus as described in Curiel, Hum. Gene Ther. (1992) 3: 147 can also be used.

  Polycationic enriched DNA linked or not linked to killed adenovirus alone (see, for example, Curiel, Hum. Gene Ther. (1992) 3: 147), ligand-linked DNA (e.g., Wu, J Biol. Chem. (1989) 264: 16985), eukaryotic delivery vehicle cells (e.g., U.S. Patent No. 5,814,482; PCT International Publication No. 95/07994; International Publication No. WO 96/17072; International Publication No. 95/30763; and WO 97/42338), and non-viral delivery vehicles and methods including, but not limited to, nuclear charge neutralization or fusion with cell membranes. Naked DNA can also be used. Representative methods for introducing naked DNA are described in PCT International Publication No. 90/11092 and US Pat. No. 5,580,859. Liposomes that can serve as gene delivery vehicles are described in US Pat. No. 5,422,120; PCT WO 95/13796; WO 94/23697; WO 91/14445; and European Patent 0 524. It is described in No.968. Further approaches are described in Philip, Mol. Cell Biol. (1994) 14: 2411 and Woffendin, Proc. Natl. Acad. Sci. (1994) 91: 1581.

  The compositions described herein may be administered as part of a sustained release formulation (ie, a formulation such as a capsule or sponge having a delayed release effect of the compound after administration). Such formulations can generally be prepared using well-known techniques, such as oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Sustained release formulations may include polypeptides, polynucleotides or antibodies dispersed in a carrier matrix and / or contained within a reservoir surrounded by a rate limiting membrane. Carriers for use in such formulations are biocompatible and can also be biodegraded, with preferred formulations providing a relatively constant level of active ingredient release. The amount of active compound in the sustained release formulation depends on the site of implantation, release rate and expected duration of release, and the nature of the condition being treated.

  The compositions of the present invention include non-pharmaceutical compositions (e.g., impurities or non-sterile compositions) and pharmaceutical compositions (i.e., compositions suitable for administration to a subject or patient or patient) that can be used to prepare unit dosage forms. A large amount of drug composition useful to the manufacturer.

  In yet another aspect of the present invention, a kit for detecting E746-A750 deficient or L858R point mutation of EGFR in a biological sample is provided. The kit includes a) a binding agent that specifically binds to an E746-A750 deletion in EGFR and / or a binding agent that specifically binds to an L858R point mutation in EGFR, and b) an E746-A750 deletion in EGFR in a sample. Or instructions for detecting L858R point mutations.

  The antibodies and peptides of the present invention may also use kits that detect E746-A750 deletion or L858R point mutations in EGFR. Such a kit may further comprise a combination of packaged reagents within a predetermined amount according to the diagnostic assay instructions. If the antibody is labeled with an enzyme, the kit contains the substrate and cofactor required by the enzyme. In addition, other additives such as stabilizers, buffers, etc. may be included. The relative amounts of the various reagents can vary widely to provide a concentration in the reagent solution that substantially optimizes the sensitivity of the assay. In particular, the reagent can be provided as a lyophilized dry powder, such as an excipient that normally provides a reagent solution of an appropriate concentration upon dissolution.

  In certain embodiments, the binding agent (eg, antibody) binds to a label moiety such as a detectable marker. One or more detectable labels can be attached to the antibody. Exemplary labeling moieties are radiopaque dyes, radiocontrast agents, fluorescent molecules, spin label molecules, enzymes, or other labeling moieties of diagnostic value, particularly in radiation or magnetic resonance imaging techniques. including.

Radiolabeled antibodies according to the present disclosure can be used for in vitro diagnostic tests. The specific activity of the antibody, its binding moiety, probe, or ligand depends on the half-life, the isotopic purity of the radiolabel, and how the label is incorporated into the biological agent. In immunological assay tests, usually the higher the specific activity, the better the sensitivity. Radioisotopes include, for example, diagnostic iodine ( ¹³¹ I or ¹²⁵ I), indium ( ¹¹¹ In), technetium ( ⁹⁹ Tc), phosphorus ( ³² P), carbon ( ¹⁴ C), and tritium ( ³ H). Or as a label comprising one of the above therapeutic isotopes.

  Biological agents labeled with fluorophores and chromophores can be prepared from standard moieties known in the art. Since antibodies and other proteins absorb light up to a wavelength of about 310 nm, a fluorescent moiety that absorbs a substantial wavelength of more than 310 nm (for example, more than 400 nm) can be selected. A variety of suitable fluorophores and chromophores are described in Stryer, Science, 162: 526 (1968) and Brand et al., Annual Review of Biochemistry, 41: 843-868 (1972), incorporated herein by reference. Has been. Antibodies can be labeled with fluorescent chromophores by conventional procedures such as those disclosed in US Pat. Nos. 3,940,475, 4,289,747, and 4,376,110, incorporated herein by reference.

  The control can be a parallel sample that provides a basis for comparison, eg, a biological sample from a healthy subject, or a biological sample from healthy tissue of the same subject. Alternatively, the control can be a predetermined reference amount or threshold amount. If the subject is being treated with a therapeutic agent and treatment progress is monitored by altered expression of the target of the invention, the control can be from a biological sample taken from the subject prior to or during the course of treatment.

  In certain embodiments, a binding agent that binds for diagnostic use of the present application is linked to an enzyme (enzyme tag) that produces a colored product upon contact of the binding agent (eg, an antibody) with a secondary binding ligand or enzyme. Intended for in vitro use. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase and glucose oxidase. In certain embodiments, secondary binding ligands are biotin and avidin or streptavidin compounds.

  The binding agents (e.g., antibodies) of the invention are optimized for use in a flow cytometry (FC) assay that determines the phosphorylation status of a target in a subject before, during, and after treatment with the therapeutic agent rain. obtain. For example, bone marrow cells or peripheral blood cells from a patient or patient can also be analyzed for flow cytometry and markers that identify various blood cell types. In this way, the nature of the activation state of the malignant cell can be specifically determined. Flow cytometry can be performed according to standard methods. See, for example, Chow et al., Cytometry (Communications in Clinical Cytometry) 46: 72-78 (2001).

  Alternatively, the antibodies of the invention can be used in immunohistochemistry (IHC) staining to detect differences in signal transduction or protein activity in normal and diseased tissues. IHC can be performed according to well-known techniques. For example, see the above-mentioned Antibodies: A Laboratory Manual.

  The peptides and antibodies of the invention may be used in other clinically relevant applications, such as bead-based multiplex type assays (e.g., IGEN, Luminex (TM) and / or Bioplex (TM) assay formats), or antibodies It can also be optimized for use in array types (such as reversed phase array applications) (see, for example, Paweletz et al., Oncogene 20 (16): 1981-89 (2001)). Accordingly, another embodiment of the present invention provides a plurality of methods for detecting a target in a biological sample, the method comprising the use of two or more binding agents of the present invention.

  In another aspect, this application relates to immunological assays for binding, purification, quantification and other common target molecule detection. Thus, in various embodiments, the amount of binding includes assay methods including, but not limited to, Western blotting, immunofluorescence, ELISA, IHC, flow cytometry, immunoprecipitation, autoradiography, scintillation counter, and chromatography. To be determined.

  The assay may be a homogeneous assay or a heterogeneous assay. In the case of a homogeneous assay, the immune response usually involves the antibody of the invention, the labeled test compound, and the sample of interest. The signal emitted by the label is directly or indirectly modified upon antibody binding to the labeled test compound. Both the immune response and its detection range are performed in the same type of solution. Immunochemical labels that can be used include free radicals, radioisotopes, fluorescent dyes, enzymes, bacteriophages, coenzymes and the like. In the case of a heterogeneous assay approach, the reagent is typically a specimen that is an antibody of the invention and is a suitable method for producing a detectable signal. Similar specimens as described above may be used. The antibody is generally immobilized on a support, such as a bead, plate or slide, and contacted with a specimen suspected of containing the antigen in the liquid phase. The support is then separated from the liquid phase and either the support phase or the liquid phase is evaluated for a detectable signal using such signal production methods. The signal is related to the presence of the test compound in the specimen. Detectable signal generation methods include the use of radioactive labels, fluorescent labels, enzyme labels and the like.

  The antibodies disclosed herein can be bound to a solid support suitable for diagnostic assays (e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene) according to known techniques such as precipitation. . In certain embodiments, the immunological assays are various types of enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs) known in the art. Detection by immunohistochemistry using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and Western blotting, dot and slot blotting, FACS analysis, etc. can also be used. Various useful immunological assay steps are described, for example, in the scientific literature such as Nakamura et al., Enzyme Immunoassays: Heterogeneous and Homogeneous Systems, Chapter 27 (1987), incorporated herein by reference. In general, detection of immune complex formation is well known in the art and can be achieved by the application of many approaches. These methods are based on detection of radioactive, fluorescent, biological or enzyme tags. Of course, as is known in the art, additional advantages may be found through the use of secondary binding ligands such as secondary antibodies or biotin / avidin ligand binding configurations. The antibody used for detection can itself bind to a detectable label and then simply detect this label. Thus, the amount of primary immune complex in the composition is determined.

  Alternatively, primary antibody binding in the primary immune complex can be detected by using a secondary binding ligand with antibody binding affinity. In such cases, the secondary binding ligand can bind to a detectable label. Secondary binding ligands are often themselves antibodies and can therefore be referred to as “secondary” antibodies. The primary immune complex is contacted with the labeled secondary binding ligand or secondary antibody under effective conditions for a time sufficient to form a secondary immune complex. The secondary immune complex removes any non-specific binding with the labeled secondary antibody or ligand by extensive washing to detect the remaining label in the secondary immune complex.

  An enzyme-linked immunosorbent assay (ELISA) is one type of binding assay. In one type of ELISA, the antibodies disclosed herein are immobilized on a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. A suspected neoplastic tissue sample is then added to the well. After binding and washing to remove non-specifically bound immune complexes, the bound target signaling protein can be detected.

  In another type of ELISA, a neoplastic tissue sample is immobilized on the well surface and then contacted with the site-specific antibody disclosed herein. After binding and washing to remove non-specifically bound immune complexes, bound antibody is detected.

  Regardless of the form of use, ELISA has certain features in common, such as coating, incubation or binding, washing to remove non-specific binding species, and detection of bound immune complexes.

  Radioimmunoassay (RIA) is an analytical technique that relies on antigen competition for antigen binding sites on antibody molecules. A standard curve consists of data collected from a series of samples each containing the same known concentration of labeled antigen and various but known concentrations of unlabeled antigen. The antigen is labeled with a radioisotope tracer. The mixture is incubated in contact with the antibody. The free antigen is then separated from the antibody and bound to the antigen. A suitable detector, such as a γ or β radiation detector, is then used to determine the proportion of either or both of the bound or released labeled antigen. This procedure is repeated for several samples containing various known concentrations of unlabeled antigen and the results are plotted as a standard graph. The bound tracer antigen ratio is plotted as a function of antigen concentration. Typically, as the total concentration of antigen increases, the relative amount of tracer antigen bound to the antibody decreases. After preparing the standard graph, the standard graph is used to determine the antigen concentration in the sample under analysis.

  In the analysis, the sample whose antigen concentration is to be determined is mixed with a known amount of tracer antigen. The tracer antigen is an antigen that is identical to a known antigen in the sample but is labeled with a suitable radioisotope. The tracer-containing sample is then incubated with the antibody. The remaining free antigen in the sample can then be measured by a detector suitable for measurement. Antigens bound to antibodies or immunosorbents can be measured in the same way. The antigen concentration of the original sample is then determined from the standard curve.

  The following examples are provided only to further illustrate the present invention and are not intended to limit the scope thereof, except as set forth in the claims appended hereto. The present invention includes modifications and variations of the methods taught herein that will be apparent to those skilled in the art.

RmAb production
New Zealand rabbits were immunized with a synthetic peptide matched to the EGFR sequence with the E746-A750del or L858R mutation. In the case of EGFR E746-A750del, the amino acid sequence of the immunogen used was CKIPVAIKTSPKANKE (SEQ ID NO: 53). In the case of the EGFR L858R mutation, the amino acid of the immunogen used was CKITDFGRAKLLGAE (SEQ ID NO: 54). Note that in both these immunogens, the EGFR sequence does not contain an N-terminal cysteine residue, but rather it is a convenient docking point for the carrier, keyhole limpet hemocyanin (KLH). Thus, the immunogenic portion of the immunogen is actually KIPVAIKTSPKANKE (SEQ ID NO: 55) for EGFR E746-A750del and KITDFGRAKLLGAE (SEQ ID NO: 56) for EGFR L858R. Rabbits that reacted positively were identified by Western blotting and preliminary IHC screening and selected for rabbit monoclonal preparation. Supernatants from newly generated clones were screened by ELISA for reactivity with immunogenic peptides.

  It is known that E746-A750del thus identified by ELISA has a specificity for EGFR or specificity for EGFR L858R point mutation, and then retains E746-A750del EGFR or EGFR L858R point mutation Cell extracts made from certain cells were examined by Western blot analysis. A panel of 6 human cancer cell lines expressing either amplified or unamplified wild type EGFR (wtEGFR), or either EGFR mutation E746-A750del or L858R was used. H3255 cell line (EGFR amplification with L858R point mutation was provided by Dr. Lewis Cantley (Harvard Medical School, Boston, Mass.) H1975 cell line (EGFR L858R point mutation) and H1650 cell line (EGFR E746_A750del) Was purchased from the American Type Culture Collection, Manassas, VA ('ATCC'). The following cell lines: HCC827 (EGFR amplification with E746-A750del), Kyse450 (human esophageal squamous cell carcinoma cell line with amplified wtEGFR) and Kyse70 (human esophageal squamous cell carcinoma cell line with wtEGFR without amplification) Was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH in Braunschweig, Germany ('DSMZ').

  For western blotting analysis, cultured cells were washed twice with cold 1 × PBS, then 1 × cell lysis buffer (20 mM Tris-HCL, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 (Sodium, sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg / mL leupeptin), and complete, mini, EDTA-free protease inhibitor cocktail (Roche) was added. . The lysate was sonicated and centrifuged at 14000 rpm for 5 minutes. Protein concentration was measured using Coomassie protein assay reagent (Pierce Chemical Co., Rockford, IL). An equal amount of total protein was resolved by 8% pre-cast Tris-Glycine gels (Invitrogen). Proteins were blotted onto nitrocellulose membranes and incubated overnight at 4 ° C. with RmAbs according to standard method protocols (see, eg, Ausubel et al., Supra). Specific binding was detected by HRP-conjugated species-specific secondary antibody and visualized using LumiGLO expression and X-ray film emission.

  As shown in FIG. 1, E746-A750del (dEGFR) RmAb detects only EGFR (E746-A750del) in HCC827 and H1650 cells, while L858R RmAb detects EGFR (L858R) in H3255 and H1975 cells. These two mutation-specific antibodies are cells of two human esophageal squamous cell carcinomas that contain the wild type sequence of exon 19 (resulting in E746-A750 deletion) and exon 21 (resulting in the L858R point mutation of EGFR) Does not react with EGFR in strains (Kyse450 and Kyse70). As expected, the control EGFR RmAb (clone 86) reacted with EGFR in all cases (see FIG. 1).

  After selection of hybridoma clones, further analysis was performed on antibodies produced by hybridomas, including immunohistochemistry of cell extracts made from the cells listed above. Finally, the clones were tested for the ability of the antibodies to specifically bind to their targets in such applications as flow cytometry and immunofluorescence. Clones that produced specific antibodies were deposited with the ATCC on April 10, 2009. A clone producing E746-A750del (dEGFR) RmAb (clone 6B6F8B10) and a clone producing EGFR (L858R) (clone 43B2E11E5B2) were assigned ATCC deposit number PTA-9151 and ATCC deposit number PTA-9152, respectively.

Immunocytochemistry Next, fluorescent immunocytochemistry analysis was performed on slides of H3255, H1975, H1650, and HCC827 cell lines using L858R, dEGFR, and control EGFR antibodies.

  In fluorescent immunocytochemistry of cells, cell lines were grown to about 70% density on 8-well chamber slides (BD, Franklin Lakes, NJ). Cells were fixed in 4% formaldehyde (Polysciences, Warrington, Pa.) In PBS for 15 minutes at room temperature, washed with PBS (10 minutes each, 3 times), then 5% normal goat serum (Sigma-Aldrich, St. Blocking was performed in a PBS solution containing 0.3% Triton X-100 (Mallinckrodt Baker, Phillipsburg, NJ) from Louis, MO for 1 hour at room temperature. Blocking solution was aspirated from the chamber and cells were incubated overnight at 4 ° C. in primary antibody diluted in PBS solution of 0.3% Triton and 1% BSA (American Bioanalytical, Natick, Mass.). Slides were washed with PBS (3 times for 10 min each), then AlexaFluor® 488-conjugated goat anti-rabbit IgG secondary antibody (Invitrogen, Carlsbad) diluted in PBS containing 0.3% Triton and 1% BSA , CA) for 1 hour at room temperature. Slides were washed in PBS as before, the chamber was removed from the slides and coverslipped with Prolong Gold anti-fade mount media (Invitrogen). Cells were imaged with a Nikon C1 confocal microscope.

  In both fluorescence immunization and immunohistochemistry (IHC) analysis, cell pellets of Kyse70 and Kyse450 cells were used as controls (Supplement: Kyse70 cells and Kyse450 were embedded in paraffin in IHC analysis. Example 3 and below. (See Figure 3).

  As shown in FIG. 2, the wtEGFR specific antibody stained all 6 cell lines regardless of the EGFR mutation status (upper row). L858R-specific antibodies only stained cancer cells with L858R point mutations (ie, H1975 and H3255 cells) (ie, specifically bound only to the cancer cells) (see FIG. 2, middle column). dEGFR-specific antibodies (ie, E746-A750del-specific antibodies) stained only cancer cells with E746_A750 mutant EGFR (ie, H1650 and HCC827 cells) (FIG. 2, bottom row).

  Thus, an L858R-specific antibody is specific for its mutant EGFR (i.e., specifically bound to an EGFR mutant with an L858R point mutation) and an EGFR containing wild-type EGFR or E746-A750 deficient It did not bind to any of the mutants. Similarly, dEGFR-specific antibodies were specific for that mutant EGFR (i.e., specifically bound to EGFR mutants, including E746-A750 deficient and have wild-type EGFR or L858R point mutations) Did not bind to any of the EGFR mutants).

Immunohistochemistry in xenografts To test the binding specificity of the rabbit monoclonal antibody described in Example 1, xenografts of human cancer cells in nude mice were prepared.

In xenografts, inoculate H3255, H1975, H1650, and HCC827 cells subcutaneously (subcutaneous injection) in the right thigh of nude (nu / nu) mice (5 × 10 ⁶ to 2 × 10 ⁷ cells / mouse) Tumors were grown to about 10 mm in diameter.

  In immunofluorescence analysis, all analyzes were performed on formalin-fixed paraffin embedded blocks. In an immunohistochemistry study, a series of 4 μm thick tissue sections were cut from TMA. Slides were dried overnight at 55 ° C., then deparaffinized in xylene and rehydrated with a series of graded ethanol concentrations. Antigen activation (boiling for 10 minutes in microwave treatment in 1 mM EDTA) was performed. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 minutes. To block nonspecific antibody binding, 5% goat serum (Sigma) solution was used, and optimally diluted primary antibody was used to cover the specimen. Slides were incubated overnight at 4 ° C. After washing 3 times with TBS-T for 5 minutes each, the slides were incubated with labeled polymer HRP anti-rabbit secondary antibody for 30 minutes at room temperature. After washing three more times with TBS-T, the slides were visualized using the substrate chromogen (Envision® + kit available from Dako). Sections were scanned at low magnification. The staining intensity as well as the positive cell rate was recorded. The staining intensity was scored from 0-3 + and the staining intensity and positive cell rate were recorded.

The staining intensity score is as follows: 0, if tumor cells are completely absent or weak staining with less than 10% staining intensity; 1+, if tumor cells are more than 10% stained weakly; 2+ Established when tumor cell staining was moderate; 3+ when tumor cell staining was strong. Tumor expression of 1+, 2+, and 3+ was interpreted as positive expression of dEGFR or L858R EGFR antibody, and non-expressing (0 score) tumors were interpreted as negative. The distribution of membrane or cytoplasmic staining was also recorded and examined at high magnification. Table 1 presents an overview of the staining scoring system.

  Figure 3 uses wild-type EGFR-specific antibody (upper row), EGFR L858R-specific antibody (middle row) and EGFR dEGFR (i.e. E746-A750del, often referred to as del722-726) specific antibody H1975 (unamplified L858R mutation), H3255 (amplified L858R mutation), H1650 (unamplified E746-A750del mutation), HCC827 (amplified E746-A750del mutation), Kyse750 (unamplified) Wild-type EGFR) and IHC stained photographs of representative samples of Kyse 450 (amplified wild-type EGFR) xenografts are presented. Because amino acid numbering begins on EGFR mutants that contain the EGFR E746-A750del signal sequence and no EGFR del722-726 signal sequence, the EGFR E746-A750del mutation is often referred to in the present and scientific literature as EGFR del722. Note that it is called -726 (ie, residues 722-726 deleted).

  As shown in FIG. 3, paraffin-embedded xenografts showed proper staining with control and mutation-specific antibodies. All cells were labeled (ie, stained or conjugated) with wtEGFR control antibody (top row of FIG. 3). The signal remained in the plasma membrane and cytoplasm, as expected by the constitutively active EGF receptor. The fluorescence intensity was proportional to the estimated EGFR expression level, that is, the expression-amplified cells (+ amp) emitted a brighter signal than the low-expression cells (−amp). Staining with mutation-specific antibodies was seen only in cancer cells, not in normal tissues, and its localization correlated with control EGFR antibody staining. The L858R antibody was highly labeled (ie, bound) only to L858R positive cells (H3255 and H1975, middle row) in H3255 xenografts with high L858R EGFR expression due to EGFR gene amplification. L858R specific antibody binding was not seen in wild type EGFR expression (Kyse450 and Kyse70) cells or defective mutant (HCC827 and H1650) cells with L858R specific antibodies. Defect specific antibodies (i.e. dEGFR specific antibodies) only labeled cells expressing EGFR deficiency (HCC827 and H1650) and the intensity was higher in HCC827 cells with EGFR amplification (Figure 3, Compare the middle two panels on the bottom row). Wild type EGFR expressing cells (ie, Kyse450 and Kyse70) and L858R mutant (H3255 and H1975) cells were not labeled with E746-A750 deficient specific antibodies (FIG. 3, bottom row).

  Note that weak staining was observed in L858R specific antibodies in HCC827 xenografts in tissue regions with high EGFR expression. This seems to be the result of cross-reaction with 43B2 antibody with high wild-type EGFR level. Similarly, weak staining (ie, binding) of EGFR E746-A750 specific (6B6F8B10) antibody was observed in H3255 and H1975 xenografts, which is due to the use of suboptimal working concentrations of this antibody. It may be due to staining.

Genotyped human tissue immunohistochemistry (IHC)
The two EGFR mutation-specific antibodies described herein (i.e., EGFR E746-A750 specific antibody and EGFR L858R specific antibody) are used for immunohistochemistry on NSCLC patient samples with EGFR genotype specification It was. Therefore, these patient samples were known for EGFR mutation status by DNA sequencing prior to being subjected to IHC analysis.

  In these tests, all analyzes were performed on formalin fixed paraffin embedded blocks. Human samples of NSCLC paraffin blocks were provided by the pathological department of Second Xiangya Hospital, Central South University (Changsha, Hunan, P.R.China). These tissues were examined with hematoxylin and eosin to confirm histopathological diagnosis and selected as appropriate specimens for further analysis. Immunohistochemistry with wild-type EGFR antibody was used to screen for EGFR positive samples (++ / +++ and +++ / +++) for molecular testing.

  In sequencing, hematoxylin and eosin stained sections of formalin-fixed paraffin-embedded tissues were examined to identify tissue regions containing at least 50% tumor cells. Examples containing less than 50% of the tumor cells or limited tumor tissue volume were excluded for non-selective screening. Genomic DNA was isolated using FormaPure kit (Agencourt Bioscience, Beverly, MA) according to the manufacturer's instructions. The exon sequence of EGFR (kinase region) was amplified with specific primers by nested polymerase chain reaction (nested PCR). The molecular type of the sample was preselected by DNA sequencing of exon 19 and exon 21 of EGFR.

FIG. 4 shows immunohistochemical staining of four representative, non-limiting, pre-molecule specific NSCLC samples with wtEGFR, E746-A750del and L858R mutant EGFR antibodies. This same IHC analysis was performed on additional pre-identification NSCLC paraffin samples and EGFR variant-specific antibodies of the invention (i.e., EGFR L858R (43B2E11E5B2) rabbit mAb and EGFR del722-726 (D6B6F8B10) with these samples. The IHC results of staining (ie binding) with rabbit mAb) were scored using the scoring system described in Table 1 above. Since keratin is present on all epithelial cells including lung cells, staining with pan-keratin specific antibody (Cell Signaling Technology, Danvers, MA) was used as a control. The gene sequences of these samples were determined before IHC analysis. Table 2 presents the score results of the IHC results compared to the gene sequencing results obtained before the IHC analysis, and the “failure” category indicates that the sample-derived DNA is too degraded for the sequence to be acquired. It shows that.

  As shown in Table 2, 5% of the samples that were IHC (+) could not be screened by sequencing (ie, they “failed”). Thus, mutant tumors may be detected by IHC where the sample DNA has been degraded or damaged to the extent that DNA sequencing is not possible and a “failure” result has been reached. 6.7% of the samples were IHC (+), but were wild type according to sequence analysis. Real-time PCR can help confirm the presence of EGFR mutations (ie, L858R or del722-726 mutations) in these samples. Finally, 15% of the samples were IHC (-) and sequence (+). This finding may be due to low expression levels of EGFR mutants or poor quality of tissue samples. In these samples, the staining with control pankeratin antibodies was weak, meaning that these tissue samples were not of good quality in IHC.

  Therefore, a 100% correlation between IHC data and EGFR mutation status data was observed in these tumor samples.

  Because the interpretation of immunohistochemical results depends on the intensity of staining in individual cancer cells, some tumor samples with low cancer cell rate mutations can be detected by IHC using mutant EGFR antibodies, but directly sequenced Then it is overlooked. In addition, this assay allows examination of paraffin blocks from small biopsy samples where extraction of high quality DNA sufficient for sequencing is difficult. Thus, this immunohistochemical assay using the two EGFR variant-specific antibodies described herein is a simple, rapid, sensitive, and reliable method of identifying specific EGFR mutations in NSCLC An assay. This immunohistochemical assay can also measure total EGFR protein concentration when wtEGFR specific antibodies are included.

  Thus, IHC-positive tumors with both wtEGFR and mutant EGFR antibodies show stronger EGFR protein expression in all xenografts and NSCLC samples, while mutant EGFR antibodies are IHC negative, but wtEGFR antibodies are positive Shows EGFR overexpression without the E746-A750del and L858R point mutations. Screening such mutant EGFR proteins in cancer (eg, lung cancer such as NSCLC, or other cancers, particularly adenocarcinoma) by immunohistochemistry may identify patients who are responsive to therapeutic agents such as gefitinib and erlotinib.

Non-selected tumors Next, IHC was performed on NSCLC tumors that had not previously been subjected to DNA sequence analysis to determine if the antibodies of the invention can be used when the genotype of the patient sample is not available. Carried out. That is, the genotype of these tumor samples was unknown.

  For these studies, paraffin-embedded tumor specimens from 340 primary NSCLC patients were screened for the presence of EGFR-deficient and EGFR L858R point mutations by IHC using a panel of four antibodies. Although these 340 patients were known to have NSCLC, the EGFR gene sequence of the NSCLC has not been determined. The antibody panel included two EGFR mutation-specific antibodies, a control wild-type EGFR-specific antibody, and a pancytokeratin-specific antibody to verify the quality of paraffin block tissue (keratins It is present in all epithelial cells, including NSCLC lung cancer cells, and binds to pancytokeratin specific antibodies).

  Two IHC results of two patients, ie, representative NLSCS tumors from CL761 and CL764, are shown in FIG. As shown in FIG. 5, the patient CL761-derived tumor sample showed positive staining with pancytokeratin-specific antibody, control wtEGFR-specific antibody and L858R-specific antibody, but negative with dEGFR-specific antibody staining. In contrast, tumor samples from patient CL764 were positive for pancytokeratin control EGFR and dEGFR antibodies but negative for L858R antibody.

  From the findings of these IHC analysis results, DNA sequence analysis of these two patient tumor samples confirmed the presence of the L858R mutation in the patient CL761 tumor and the presence of the E746-A750 deletion in the patient CL764 tumor.

  IHC was performed on a total of 340 NSCLC samples from patients with unknown genotypes (i.e., samples for which EGFR mutations had not been previously identified by DNA analysis), using the scoring criteria listed in Table 1. Scored. These 340 NSCLC samples were classified into subgroups of NSCLC pathological diagnosis: adenocarcinoma (AC), squamous cell carcinoma (SCC), and large cell carcinoma (LCC).

The results of these IHC analyzes are presented in Table 3.

  As shown in Table 3, 24 cases (7.1%) scored positive for E746-A750 deficient antibody, and 28 cases (8.2%) scored positive for L858R antibody. Interestingly, as shown in Table 3, the NSCLC subgroup with the highest number of either EGFR L858R or dEGFR (ie, E746-A750) mutations was adenocarcinoma cells. The adenocarcinoma in Table 3 (and Table 4 below) is NSCLC, but adenocarcinoma is colon cancer, breast cancer, cervical cancer, pancreatic cancer (e.g., most pancreatic cancer is ductal adenocarcinoma), prostate cancer, stomach cancer, It is also expressed in cancers including but not limited to esophageal cancer.

  In addition, 52 patients (15.3%) were positive for both EGFR mutation-specific antibodies. 84.6% of mutated EGFR positive cases showed moderate to strong staining with control wtEGFR-specific antibody and presented above that wild-type EGFR-specific antibody is inappropriate in detecting tumor samples with EGFR mutation It was confirmed.

To confirm IHC results, direct DNA sequence analysis of the EGFR gene (exons 19 and 21) was performed on tumor specimens including whole adenocarcinoma samples from 244 patients and a small number of squamous cell carcinoma and large cell carcinoma samples. These results are presented in Table 4 below. Note that the “failure” category indicates that the DNA from these samples was too damaged or degraded to obtain proper sequencing.

  As noted, 51 of 244 patients' tumor sample DNA was too degraded for sequencing.

  As shown in Table 4, all EGFR L858R mutations were found in adenocarcinoma and 23 of 24 EGFR E746-A750del mutations were found in adenocarcinoma. Thus, the IHC assays described herein are very useful for detecting NSCLC (or another tumor type) that falls into the adenocarcinoma subgroup.

In addition, the genotype of all samples (9 samples total) that were positive for control EGFR antibody but differed between IHC and direct DNA sequencing results was identified by a system based on Sequenom mass spectrometry (MS) . This technique has been reported to be more accurate than direct DNA sequencing in genotyping low quality DNA obtained from formalin-fixed paraffin-embedded tissue (FFPET) (Jaremko et al., Hum Mutat 25: 232-238, 2005 ). Table 5 shows the MS sequencing results of these nine tumor samples that showed inconsistencies between IHC staining and direct DNA sequencing.

From the results shown in Table 5, the different analytical methods used (ie, IHC staining, direct DNA sequencing, and MS sequencing) were correlated with the results presented in Table 6 below.

  As shown in Table 6, a higher correlation was found between Sequenom and IHC results than between direct DNA sequencing and IHC results. This finding suggests that EGFR mutation-specific IHC may be more accurate than EGFR direct DNA sequencing.

  In general, detection of these two EGFR mutations by IHC was confirmed in 47 of 52 cases by either direct DNA sequencing or Sequenom analysis. Overall, IHC assays using mutation-specific antibodies were found to be 92% sensitive and 99% specific. DNA sequence analysis further identified 5 cases containing EGFR mutations that were negative in IHC using EGFR variant specific antibodies. However, these samples were negative for IHC by either control EGFR or pancytokeratin staining, suggesting that these samples were too poor for IHC. This suggests that PCR amplification and DNA sequencing can improve mutation detection for cases involving poorly conserved tissues.

Sequence analysis The cDNA and amino acid sequences of the EGFR E746-A750del (6B6F8B10 (often referred to as D6B6F8B10 clone or simply 6B6 clone)) rabbit monoclonal antibody heavy chain determined using the above method are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. . The cDNA and amino acid sequences of the EGFR E746-A750del (clone 6B6F8B10) rabbit monoclonal antibody light chain are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The cDNA and amino acid sequences of the EGFR L858R (clone 43B2E11E5B2) rabbit monoclonal antibody heavy chain are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The cDNA and amino acid sequences of the EGFR L858R (43B2E11E5B2) rabbit monoclonal antibody light chain are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

  Complementarity determining regions (CDRs) and framework regions (FWRs) as defined by the Kabat criterion are the Wu and Kabat method (Wu, TT and Kabat, EA (1970) J. Exp. Med., 132, 211-250 ) Using the full-length heavy and light chain sequences of EGFR del722-726 (6B6F8B10) and EGFR L858R (43B2E11E5B2) rabbit monoclonal antibodies.

The EGFR E746-A750del (6B6F8B10) rabbit mAb region was determined to have the following amino acid sequence:
Heavy chain complementarity determining region (CDR) and framework region (FWR)

CDR1: FFSSNNDWMC (SEQ ID NO: 9)
CDR2: CIYGGSSIGTNYAGWAKG (SEQ ID NO: 10)
CDR3: DLANL (SEQ ID NO: 11)

FWR1: HCQSLEESGGGLVKPGASLTLTCTASG (SEQ ID NO: 12)
FWR2: WVRQAPGKGLEWIA (SEQ ID NO: 13)
FWR3: RFTISRTSSTTVALQMTSLTVADTATYFCTR (SEQ ID NO: 14)
FWR4: WGPGTLVSVSS (SEQ ID NO: 15)

Light chain complementarity determining region (CDR) and framework region (FWR): defined by Kabat criteria

CDR1: QSSQSVYSDWLS (SEQ ID NO: 16)
CDR2: EASKLAS (SEQ ID NO: 17)
CDR3: LASYDCTRADCLA (SEQ ID NO: 18)

FWR1: AQVLTQTPSSVSAAVGGTVTINC (SEQ ID NO: 19)
FWR2: WYQQKGGQPPRQLIY (SEQ ID NO: 20)
FWR3: GVPSRFSGSGSGTQFTLTINDVQCDDAATYYC (SEQ ID NO: 21)
FWR4: FGGGTEVVVR (SEQ ID NO: 22)

3197 EGFR L858R (43B2E11E5B2) rabbit mAb region was determined to have the following amino acid sequence:
3197 EGFR L858R (43B2E11E5B2) rabbit mAb

Heavy chain complementarity determining regions (CDRs) and framework regions (FWRs): defined by Kabat criteria
CDR1: FSLNTYGVS (SEQ ID NO: 23)
CDR2: YIFTDGQTYYASWAKG (SEQ ID NO: 24)
CDR3: VDI (SEQ ID NO: 25)

FWR1: QCQSVEESGGRLVTPGTPLTLTCTVSG (SEQ ID NO: 26)
FWR2: WVRQAPGKGLEWIG (SEQ ID NO: 27)
FWR3: RFTISKTSSTTVDLKITSPTTEDTATYFCAS (SEQ ID NO: 28)
FWR4: WGPGTPVTVSS (SEQ ID NO: 29)

Light chain complementarity determining region (CDR) and framework region (FWR): defined by Kabat criteria

CDR1: QSSPSVYSNYLS (SEQ ID NO: 30)
CDR2: DASHLAS (SEQ ID NO: 31)
CDR3: LGSYDCSSVDCHA (SEQ ID NO: 32)

FWR1: AQVLTQTPSPVSAAVGSTVTIKC (SEQ ID NO: 33)
FWR2: WYQQKSGQPPKQLIY (SEQ ID NO: 34)
FWR3: GVPSRFSGSGSGTQFTLTISGVQCDDAATYYC (SEQ ID NO: 35)
FWR4: FGGGTEVVVK (SEQ ID NO: 36)

Heavy and light chain VDJ and VJ configurations were further identified.
The heavy and light chain VDJ and VJ configurations of the EGFR E746-A750del (6B6F8B10) rabbit monoclonal antibody were identified as follows.
Heavy chain VDJ configuration.

The V region is VH1a3.
HCQSLEESGGGLVKPGASLTLTCTASGFSFSNNDWMCWVRQAPGKGLEWICIYGGSSIGTNYAGWAKGRFTISRTSSTTVALQMTSLTVADTATYFCTR (SEQ ID NO: 37)

The D region is too short for identification.
DLA (SEQ ID NO: 38)

The J region is JH4.
NLWGPGTLVSVSS (SEQ ID NO: 39)

Light chain VJ configuration.

The V region is as follows.
MDMRAPTQLLGLLLLWLPGATFAQVLTQTPSSVSAAVGGTVTINCQSSQSVYSDWLSWYQQKGGQPPRQLIYEASKLASGVPSRFSGSGSGTQFTLTINDVQCDDAATYYCLASYDCTRADCL (SEQ ID NO: 40)

The J region is JK2.
AFGGGTEVVVR (SEQ ID NO: 41)

The heavy and light chain VDJ and VJ configurations of the EGFR L858R (43B2E11E5B2) rabbit monoclonal antibody were determined as follows.
Heavy chain VDJ configuration.

The V-gene is VH1a1.
QCQSVEESGGRLVTPGTPLTLTCTVSGFSLNTYGVSWVRQAPGKGLEWIG
YIFTDGQTYYASWAKGRFTISKTSSTTVDLKITSPTTEDTATYFCAS (SEQ ID NO: 42)

The D-gene is too short for identification.
VDI (SEQ ID NO: 43)

The J region is JH4.
WGPGTPVTVSS (SEQ ID NO: 44)

Light chain VJ configuration.
V region:
MDMRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAVGSTVTIKCQSSPS
VYSNYLSWYQQKSGQPPKQLIYDASHLASGVPSRFSGSGSGTQFTLTISG
VQCDDAATYYCLGSYDCSSVDCH (SEQ ID NO: 45)

The J region is JK2.
AFGGGTEVVVK (SEQ ID NO: 46)

Epitope mapping by phage display
ELISA plates were coated with 100 μg / mL of antibody in 0.1 M NaHCO ₃ (pH 8.6). 100 μL of diluted mAb sample was added to each well and incubated overnight at 4 ° C. with gentle agitation. Plates were washed and incubated for 1 hour at 4 ° C. in blocking buffer (5 mg / L BSA, 0.02% NaN ₃ in 0.1M NaHCO ₃ solution, pH 8.6), then washed rapidly 6 times with TBST. Phage display libraries Ph.D.-7 and Ph.D-12 were purchased from New England BioLabs (Ipswich, Mass.). The library was diluted 2 × 10 ¹¹ with 100 μL TBST, added to the plate and incubated for 60 minutes at room temperature with gentle agitation. The plate was then washed 10 times with TBST. Bound phage was eluted with 100 μL 0.2 M glycine-HCl (pH 2.2), 1 mg / mL BSA for 10 minutes. The eluate was neutralized with 15 μL 1M Tris-HCl (pH 9.1). The eluted phage was amplified in ER2738 culture at 37 ° C. for 4 and a half hours with vigorous stirring. The amplified phage was centrifuged at 10,000 rpm for 10 minutes at 4 ° C, then 80% of the supernatant was transferred to an unused tube and 1/6 volume of PEG / NaCl [20% (w / v) PEG-8000, 2.5M NaCl ] Was added overnight at 4 ° C. to precipitate the phage. The phage was isolated by centrifugation at 10,000 × g for 20 minutes at 4 ° C., and the remaining cell debris was pelleted. The supernatant was transferred to an unused microcentury fuse tube and reprecipitated with 1/6 volume of PEG / NaCl on ice for 60 minutes. Phages were isolated by centrifugation at 4 ° C. for 10 minutes and resuspended in 200 μL TBS, 0.02% NaN ₃ . The isolated phage was centrifuged for 1 minute to pellet any residue in the soluble material. The supernatant was transferred to an unused tube and the amplified phage was titrated on LB medium plates containing IPTG and X-gal. The second and third round biopanning protocols were the same as the first round.

The immunogen used for EGFR L858R and EGFR-deficient monoclonal antibody was a short peptide of 15 and 16 amino acids in length (see Example 1). These peptides bind to keyhole limpet hemocyanin (KLH), a high molecular weight protein, a complex widely used as a carrier protein in antibody production because it provides superior immunogenicity to the bound antigen. These immunogens bind strongly to each monoclonal antibody. Two different phage display libraries available from New England BioLabs (Ipswich, MA) were used to determine the 5-6 amino acid core epitope. The PhD7 library is the most characterized and encodes most, if not all, possible 7-residue sequences. With this library, fewer clones are extracted, which are clones that have a strong binding affinity compared to the other library PhD12. PhD12 encodes a 12-residue sequence and eliminates more clones that may form multiple weak bonds. EGFR E749-A750del rabbit mAb that screened both PhD7 and PhD12 suggested that “TSP” (Table 7) is a potentially important region within the immunogen. The “TSP” site is directly adjacent to the missing site. These experiments can be verified by peptide ELISA.

Phage display using EGFR L858R rabbit mAb identified a clear consensus sequence “TDXGR” using the PhD12 library. These data are summarized in Table 8. These data can vary by peptide ELISA.

Table 9 presents a summary of the consensus sequence of two EGFR variant specific antibodies.

  To validate the consensus sequence obtained from the phage display library (Table 9), intra-antigen residues can be mutated to alanine, and changes important for binding can be analyzed to perform an alanine scan. Both EGFR mutant antibodies were immunized with a short peptide sequence ranging from 15 to 16 amino acids. In these antibodies, epitope mapping can be performed by peptide ELISA using mutated versions of these immunogens.

Claims (14)

A binding agent that specifically binds to an epidermal growth factor receptor (EGFR) molecule having a point mutation in which leucine at position 858 is replaced with arginine, wherein the binding agent is an antibody,
With heavy chain CDR1, CDR2, and CDR3 having SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively, or one or several conservative substitutions, deletions or additions of amino acids that do not alter the specific binding Comprising heavy chains CDR1, CDR2, and CDR3, and
With light chain CDR1, CDR2, and CDR3 having SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively, or with one or several conservative substitutions, deletions or additions of amino acids that do not alter the specific binding A binding agent comprising light chains CDR1, CDR2, and CDR3. A binding agent that specifically binds to an epidermal growth factor receptor (EGFR) molecule having a point mutation in which leucine at position 858 is replaced with arginine, wherein the binding agent is an antibody,
With heavy chain CDR1, CDR2, and CDR3 having SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, respectively, or one or several conservative substitutions, deletions or additions of amino acids that do not alter the specific binding Comprising heavy chains CDR1, CDR2, and CDR3, and
With light chain CDR1, CDR2, and CDR3 having SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, respectively, or one or several conservative substitutions, deletions or additions of amino acids that do not alter the specific binding A binding agent comprising light chains CDR1, CDR2, and CDR3.   The binding agent according to claim 1 or 2, wherein the EGFR molecule is derived from a human.   The binding agent according to claim 1 or 2, wherein the binding agent is a rabbit monoclonal antibody.   2. The binding agent of claim 1, wherein the binding agent specifically binds to an epitope comprising an amino acid sequence comprising a threonine-serine-proline sequence.   The binding agent according to claim 2, wherein the binding agent specifically binds to an epitope comprising an amino acid sequence comprising a threonine-aspartic acid-X-glycine-arginine sequence, wherein X is any amino acid residue. .   A polynucleotide encoding the binding agent according to claim 1 or 2.   A vector comprising the polynucleotide according to claim 7.   A method for identifying cancer in which targeted therapy for abnormal expression of EGFR molecule is effective, (a) contacting a cancer-derived biological sample with the binding agent according to claim 1 or 2 to obtain a binding amount; and (b ) Including a step of comparing the result of the step (a) with a binding amount obtained by bringing a biological sample derived from a healthy individual into contact with the binding agent according to claim 1 or 2, wherein the binding amount derived from the cancer is A method for identifying cancer, characterized by showing that the therapy responds well to the cancer when the amount of binding derived from the healthy individual is exceeded.   10. The amount of binding determined using an assay method selected from the group consisting of Western blot, immunofluorescence, ELISA, IHC, flow cytometry, immunoprecipitation, autoradiography, scintillation counter, and chromatography. the method of.   The method of claim 9, wherein the cancer is from a human patient.   10. The method of claim 9, wherein the cancer is non-small cell lung cancer (NSCLC).   The method of claim 9, wherein the cancer is adenocarcinoma or squamous cell carcinoma.   10. The method of claim 9, wherein the cancer is a tissue type selected from the group consisting of lung cancer, colon cancer, breast cancer, cervical cancer, pancreatic cancer, prostate cancer, gastric cancer, and esophageal cancer.

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